Recombinant chimeric bovine/human parainfluenza virus 3 expressing RSV G and its use

ABSTRACT

Recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3) vectors expressing Respiratory Syncytial Virus (RSV) G protein or a recombinant RSV G protein, as well as methods of their use and manufacture, are provided. The rB/HPIV3 vector comprises a genome comprising a heterologous gene encoding the RSV G protein or the recombinant RSV G protein. Nucleic acid molecules comprising the sequence of the genome or antigenome of the disclosed rB/HPIV3 vectors are also provided. The disclosed rB/HPIV3 vectors can be used, for example, to induce an immune response to RSV and HPIV3 in a subject.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No. PCT/US2018/034848, filed on May 29, 2018, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Application No. 62/512,111, filed May 29, 2017. The provisional application is herein incorporated by reference in its entirety.

FIELD

This disclosure relates to recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3) vectors expressing Respiratory Syncytial Virus (RSV) G protein, or a recombinant RSV G protein, and use of the rB/HPIV3 vector, for example, to induce an immune response to RSV and HPIV3 in a subject.

PARTIES TO A JOINT RESEARCH AGREEMENT

This invention was made under Public Health Service Cooperative Research and Development Agreement (PHS-CRADA) No. 2013-0810 between the National Institute of Allergy and Infectious Disease at the National Institutes of Health and Sanofi Pasteur, Inc.

BACKGROUND

RSV is an enveloped non-segmented negative-strand RNA virus in the family Pneumoviridae, genus Orthopneumovirus. It is the most common cause of bronchiolitis and pneumonia among children in their first year of life. RSV also causes repeated infections including severe lower respiratory tract disease, which may occur at any age, especially among the elderly or those with compromised cardiac, pulmonary, or immune systems. Passive immunization currently is used to prevent severe illness caused by RSV infection, especially in infants with prematurity, bronchopulmonary dysplasia, or congenital heart disease. Despite the burden of RSV infection in certain populations, development of an effective RSV vaccine remains elusive.

Parainfluenza viruses (PIV) are closely related enveloped non-segmented negative-strand RNA viruses that belong to the closely related family Paramyxoviridae. PIVs include members of the genus Respirovirus (including PIV1, PIV3, Sendai virus) and Rubulavirus (including PIV2, PIV4, PIV5). The human parainfluenza viruses (HPIVs, serotypes 1, 2, and 3) are second only to RSV in causing severe respiratory infections in infants and children worldwide, with HPIV3 being the most relevant of the HPIVs in terms of disease impact. The HPIV3 genome is approximately 15.5 kb, with a gene order of 3′-N-P-M-F-HN-L. Each gene encodes a separate mRNA that encodes a major protein: N, nucleoprotein; P, phosphoprotein; M, matrix protein; F, fusion glycoprotein; HN, hemagglutinin-neuramindase glycoprotein; L, large polymerase protein, with the P gene containing additional open reading frames encoding the accessory C and V proteins. Similar to RSV, development of an effective HPIV vaccine remains elusive.

Major challenges to developing pediatric vaccines against RSV and HPIV3 include the immaturity of the immune system during infancy, immune-suppression by maternal antibodies, inefficient immune protection at the superficial epithelium of the respiratory tract, and vaccine-induced enhanced disease that has been observed in studies with inactivated or subunit RSV and HPIV3 vaccines in virus-naïve recipients. Further, prior studies of a live-attenuated rB/HPIV3 vector expressing an RSV antigen (RSV F protein) revealed disappointing immunogenicity to RSV that was deemed insufficient for vaccine use.

Thus, despite substantial effort, a need remains for a safe and effective immunogen that induces a protective immune response to RSV and HPIV3, particularly in pediatric subjects.

SUMMARY

Recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3) vectors expressing RSV G or variants thereof (“rB/HPIV3-RSV G” vectors) are provided herein. The disclosed rB/HPIV3-RSV G vectors comprise a genome comprising, in a 3′-to-5′ order, a 3′ leader region, a BPIV3 N gene, a heterologous gene, BPIV3 P and M genes, HPIV3 F and HN genes, a BPIV3 L gene, and a 5′ trailer region. The heterologous gene encodes one of: (a) a RSV G protein comprising an RSV G ectodomain, transmembrane domain, and cytoplasmic tail; (b) a recombinant RSV G protein comprising a RSV G ectodomain, a BPIV3 HN transmembrane domain, and a BPIV3 HN cytoplasmic tail; (c) a recombinant RSV G protein comprising a RSV G ectodomain, a HPIV3 HN transmembrane domain, and a HPIV3 HN cytoplasmic tail; or (d) a recombinant RSV G protein comprising a RSV G ectodomain, a HPIV1 HN transmembrane domain, and a HPIV1 HN cytoplasmic tail. The HPIV3 HN gene encodes a HPIV3 HN protein comprising 263T and 370P amino acid assignments. The rB/HPIV3 vectors disclosed herein are infectious, attenuated, and self-replicating, and can be used to induce an immune response to RSV and HPIV3.

In some embodiments, the heterologous gene encoding the wild-type (wt) RSV G protein or recombinant RSV G protein can be codon-optimized for expression in human cells.

Also provided herein are methods and compositions related to the expression of the disclosed viruses. For example, isolated polynucleotide molecules that include a nucleic acid sequence encoding the genome or antigenome of the described viruses are disclosed.

Immunogenic compositions including the rB/HPIV3-RSV G are also provided. The compositions can further include an adjuvant. Methods of eliciting an immune response in a subject by administering an effective amount of a disclosed rB/HPIV3-RSV G to the subject are also disclosed. In some embodiments, the subject is a human subject, for example, a human subject between 1 and 6 months of age, or between 1 and 12 months of age, or between 1 and 18 months of age, or older.

The foregoing and other features and advantages of this disclosure will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C. Map of the rB/HPIV3 genome and added RSV G gene of rB/HPIV3-RSV G vectors, and diagrams and features of modified RSV G proteins included in the vectors. (FIG. 1A) rB/HPIV3 gene map and G protein diagrams. The rB/HPIV3 gene map is shown at the top, and the second line shows the details of the sequence flanking the RSV strain A2 G gene inserted at the second gene position, between the vector N and P genes, using the indicated AscI sites (underlined). N GE: gene end signal from the BPIV3 N gene: P GS: gene start signal from the BPIV3 P gene; M48: AUG codon 48 of the RSV G open reading frame (ORF) that can serve as an alternative translational start site; CX3C (G amino acids 182-186): fractalkine motif, conserved among RSV strains; the TGA stop codon of the G ORF is emboldened and underlined. An additional TAG stop codon is underlined with *** and was included in those G protein inserts indicated with ***: this trinucleotide was added when needed to adjust the sequence length to conform to the “rule of six” (see Kolakofsky Virology 498:94-98, 2016). Several diagrams of RSV G protein structures are shown, including: (i) wt G, unmodified wild-type RSV G; (ii) mG, expressing only the transmembrane form of RSV G (mG) due to the M48I mutation that ablates expression of secreted G (sG); (iii) sG, expressing only sG, due to deletion of the first 47 codons, so that the ORF begins with the M48 codon; (iv) G_B3CT, with the cytoplasmic tail (CT) of RSV G replaced by the CT of BPIV3 HN; (v) G_B3TMCT, with the transmembrane domain (TM) and CT of RSV G replaced by the TM and CT of BPIV3 HN; (vi) G_dCX3C, with the C186R mutation ablating the CX3C motif (yielding CX3R); (vii) G_wCX4C, with the addition of A186 to ablate the CX3C motif (yielding CX3AC); (viii) G_dCX3C_B3CT, with the C186R mutation ablating the CX3C motif, and bearing the CT of BPIV3 HN; (ix) G_dCX3C_B3TMCT, with the C186R mutation ablating the CX3C motif, and bearing the TM and CT of BPIV3 HN; (x) wt G/GS-opt, GenScript codon-optimized wt G. (FIG. 1B) Amino acid sequences of the TM and CT domains of wt RSV G protein (top); and the following modified versions of RSV G: mG, sG, G_B3CT, and G_B3TMCT; and the wt BPIV3 HN (strain Kansas), and wt HPIV3 HN (strain JS). The sequences were aligned according to the beginning of the G ectodomain at amino acid 66. The presumed CT, TM, and ectodomains are demarcated with dashed lines, and BPIV3 HN sequences are boxed. Note that the N-terminus of sG is that of the primary translation product, which subsequently gets trimmed proteolytically to yield a predominant N-terminus at N66, or a secondary N-terminus at I75. (FIG. 1C) Sequence of the unmodified CX3C motif of wt G protein (top line, showing protein (SEQ ID NO: 41) and nucleotide (SEQ ID NO: 42) sequence), the dCX3C version in which the CX3C motif is disrupted by a C186R missense mutation (middle line, showing protein (SEQ ID NO: 43) and nucleotide (SEQ ID NO: 44) sequence), and the wCX4C version in which the CX3C motif is disrupted by the addition of an alanine codon following codon 185 (bottom line, showing protein (SEQ ID NO: 45) and nucleotide (SEQ ID NO: 46) sequence). The conserved cysteine residues are in bold; mutated nucleotides in dCX3C and wCX4C are underlined.

FIGS. 2A-2D. Western blot analysis of modified RSV G proteins expressed in Vero cells from the rB/HPIV3-RSV G vectors. Vero cells were infected with the indicated rB/HPIV3-RSV G vector or wt RSV at an MOI of 10 TCID₅₀ or 3 PFU per cell, respectively. At 24 hours post-infection (h.p.i.), the cells were harvested for analysis, and at 48 h.p.i., the overlying cell culture medium supernatants from duplicate cultures were harvested for analysis. The cells and medium supernatants were analyzed separately by gel electrophoresis under denaturing and reducing conditions followed by Western blotting with antisera raised separately against RSV and HPIV3, followed by secondary antibodies conjugated with infrared fluorescent dyes, and the relative levels of the large predominant band of fully glycosylated, 90-120 kDa RSV G protein were quantified densitometric analysis using an Odyssey imaging system (LiCor). (FIG. 2A) Western blots of proteins from infected cells. Top panel: the bars to the left indicate fully and partially glycosylated forms of RSV G (upper and lower bars, respectively), lane 11 shows in addition the RSV N, P, M proteins expressed by wt RSV. Middle panel: BPIV3 N protein. Bottom panel: GAPDH protein used as loading control. (FIG. 2B) Relative levels of fully glycosylated (90-120 kDa) RSV G quantified from the experiment in part A by densitometry using the LiCor Image Studio software calibrated using the GAPDH signal and normalized to wt G (lane 2) set as 1.0. (FIG. 2C) Western blot of G protein in cell culture medium supernatants. (FIG. 2D) Relative levels of fully glycosylated G protein from FIG. 2C, normalized to wt G.

FIGS. 3A and 3B. Western blot analysis of RSV G expression from rB/HPIV3-RSV G vectors in LLC-MK2 cells. LLC-MK2 cells were infected with the indicated rB/HPIV3-RSV G constructs or wt RSV as described in FIG. 2A. Cells were harvested at 24 h.p.i. processed, and subjected to by Western blot analysis with antisera raised separately against RSV and HPIV3 as described in FIGS. 2A and 2B. (FIG. 3A) Western blot showing expression of RSV G (top panel), BPIV3 N (middle panel), and GAPDH (bottom panel) as a loading control. (FIG. 3B) Relative levels of fully glycosylated (90-120 kDa) RSV G quantified from FIG. 3A and normalized to wt G (lane 2) as 1.0.

FIGS. 4A and 4B. Double-staining plaque assays characterizing expression of modified forms of RSV G protein from rB/HPIV3-RSV G. rB/HPIV3 constructs expressing the indicated modified G proteins were inoculated in 10-fold dilution series on Vero cell monolayers in 24-well plates and incubated under methyl cellulose overlay for 6 days. The monolayers were fixed with 80% methanol and analyzed with the indicated primary antibodies followed by secondary antibodies conjugated to infrared dye. (FIG. 4A) Images of plaques that were probed with rabbit antisera raised separately against HPIV3 and RSV, and a goat hyperimmune serum to RSV (ab20531, Abcam): HPIV3 antigens alone were visualized as green, RSV G protein alone as red, and co-expression as yellow. (FIG. 4B) Images of plaques that were probed with the same HPIV3-specific rabbit hyperimmune serum and RSV G mAb 131-2G specific to the CX3C domain: HPIV3 antigens alone were visualized as green, RSV G containing the 131-2G epitope alone was red, and co-expression was yellow.

FIGS. 5A and 5B. Quantification of RSV G packaging efficiency by Western blot analysis of purified rB/HPIV3-RSV G particles. LLC-MK2 and Vero cells were infected with, respectively, 0.01 TCID₅₀ of the indicated rB/HPIV3-RSV G construct or 0.1 PFU of wt RSV. The cultures were harvested on day 4 and clarified cell culture medium supernatants were subjected to centrifugation on discontinuous 30%/60% w/v sucrose gradients. Purified virions were harvested from the sucrose interface and pelleted by centrifugation. Approximately 4 g of each virus were subjected to gel electrophoresis under denaturing and reducing conditions. Western blots were prepared and analyzed with antisera raised separately against RSV and HPIV3. (FIG. 5A) Western blot of empty rB/HPIV3 vector (lane 1), vector expressing wt RSV G (lane 2), vectors expressing the indicated modified forms of RSV G (lanes 3-5), and wt RSV (lane 6). The upper panel was probed with polyclonal RSV antibodies, showing the fully-glycosylated 90-120 kDa form of G, evident in lane 5, and RSV structural proteins N, P, M, and G proteins including a smaller, broad band of G (˜48-62 kDa) often observed with Vero cells (lane 6) as previously described (Corry et al. J. Virol. 90:1311-1320, 2015). The bottom panel shows the BPIV3 N protein. (FIG. 5B) RSV G packaging efficiency based on quantification of FIG. 5A and normalized to wt G (lane 2) as 1.0.

FIGS. 6A-6L. Imaging of packaged RSV G in virions by transmission electron microscopy (TEM) with immune-gold labeling. Purified virions from the preparations described in FIG. 5 were incubated with G-specific mouse MAb 131-2G (specific to the CX3C domain) and polyclonal goat anti-mouse secondary antibodies conjugated with 10 nm gold particles. Selected representative virion images are shown for: wt RSV (FIGS. 6A and 6B), rB/HPIV3 vector expressing wt RSV G (FIGS. 6C and 6D), rB/HPIV3 vector expressing only sG (FIGS. 6E and 6F), rB/HPIV3 vector expressing chimeric RSV G with CT of BPIV3 HN (FIGS. 6G and 6H), rBHPIV3 vector expressing chimeric RSV G with TM and CT of BPIV3 HN (FIGS. I and 6J), and empty rB/HPIV3 vector (FIGS. 6K and 6L).

FIGS. 7A and 7B. Replication of rB/HPIV3-RSV G vectors in the upper and lower respiratory tracts of hamsters. Hamsters in groups of six were infected intranasally (IN) with 10⁵ TCID₅₀ of vector or 10⁶ PFU of wt RSV. Nasal turbinates and lungs were collected on day 5 post-immunization and homogenized. Titers of vectors in nasal turbinates (FIG. 7A) and lungs (FIG. 7B) were determined by TCID₅₀ hemadsorption assays on LLC-MK2 cells. The limit of detection of TCID₅₀ is indicated as dashed line. Mean titer and standard error of the mean (SEM) are shown as horizontal line with error bars. The statistical significance of differences in mean titers among all groups in the present study was analyzed using one-way ANOVA followed by Tukey-Kramer test. The mean titers of any two groups designated with a same letter (A, or B) are not statistically different.

FIGS. 8A-8E. RSV- and HPIV3-neutralizing serum antibody titers induced by rB/HPIV3-RSV G vectors and wt RSV in immunized hamsters. Hamsters in groups of six were infected IN by 10⁵ TCID₅₀ of the indicated rB/HPIV3-RSV G vector or 10⁶PFU of wt RSV. Sera were collected on day 28 post-immunization. The serum neutralization titers were determined by 60% plaque reduction neutralization assay (PRNT₆₀) on Vero cell monolayers. For comparison, sera from a group of hamsters immunized with the same dose of vector expressing unmodified wt RSV F (from a separate experiment performed in essentially the same way) were included in RSV neutralization assays. Neutralization titers against: wt RSV, with complement (FIG. 8A), HPIV3, with complement (FIG. 8B), RSV B1, of subgroup B, with complement (FIG. 8C), RSV CX3C mutant (CWAIS), with complement (FIG. 8D), and wt RSV, without complement (FIG. 8E), are shown. The limit of detection is indicated with a dashed line. Each dot represents the titer of an individual animal. The bars indicate the mean titers of the groups; and error bars represent the SEM. The statistical significance of differences in mean titers among all groups in each assay was analyzed using one-way ANOVA followed by Tukey-Kramer test. Mean titers designated with the same letter (A, B, C or D) are not statistically different. In FIG. 8A, a Student t-test was carried out between the two indicated groups (2 and 12), with the significance of the difference shown as a P value.

FIGS. 9A-9D. Protection of immunized hamsters against wt RSV A2 challenge. Hamsters in groups of six were immunized IN with 10⁵ TCID₅₀ of the indicated rB/HPIV3-RSV G vector or 10⁶PFU of wt RSV as described in FIG. 8 and 31 days later were challenged IN by 10⁶ PFU of wt RSV. On day 3 post-challenge, animals were sacrificed and viral titers were determined in homogenates of the (FIG. 9A) nasal turbinates (NT) and (FIG. 9B) lungs by plaque assay in Vero cells. The limit of detection is indicated with a dashed line. Each dot represents the titer of an individual animal. Mean titers are indicated as short horizontal bars. The statistical significance of differences in mean titers among all groups was analyzed using one-way ANOVA followed by Tukey-Kramer test. Mean titers of groups designated with a same letter (A, B, C or D) are not statistically different. In FIGS. 9C and 9D, the RSV challenge virus titers for individual animals in the nasal turbinates (FIG. 9C) and lungs (FIG. 9D) were plotted versus the corresponding titers of complement-dependent serum RSV-neutralizing antibodies collected 28 days following vector immunization (from FIG. 8A). Pearson's linear regression model (statistical software Prism 7.0) was used to show the correlation of the neutralization titers (Log 2PRNT60) and viral titer (Log 10 PFU/g), shown by solid lines. The R squared values were 0.45 for NT and 0.51 for lungs: the R squared value indicates how well the data fit into the model (values range from 0 to 1, with a greater value indicating increased fit). The dotted lines indicate 95% confidence intervals.

FIGS. 10A-10D. Expression of G protein from a B/HPIV3 vector bearing a G ORF that was codon-optimized for human translation (construct (x) in FIG. 1A, or wt G/GS-opt). The accumulation of G protein in LLC-MK2 and Vero cells infected with rB/HPIV3 vectors expressing wt G or wt G/GS-opt was evaluated by Western blot analysis (FIGS. 10A and 10B). The LLC-MK2 (10A) and Vero (10B) cells were infected with the indicated rB/HPIV3-RSV-G vector or wt RSV at an MOI of 10 TCID₅₀ or 3 PFU per cell, respectively. At 24 hours post-infection, the cells were harvested and lysates prepared and analyzed by gel electrophoresis under denaturing and reducing conditions followed by Western blotting with antisera raised against RSV, followed by secondary antibodies conjugated with infrared fluorescent dyes, and the bound antibodies were visualized using an Odyssey imaging system (LiCor). Quantification of band intensities in Western blot analysis indicated that there was 2.3-fold increase of RSV G expression by codon-optimization in LLC-MK2 cells (FIG. 10C) and 2.2-fold increase in Vero cells (FIG. 10D).

FIG. 11 RSV-neutralizing serum antibody titers induced by rB/HPIV3-RSV-G vectors expressing codon-optimized or non-optimized G ORFs in immunized hamsters. Hamsters in groups of nine were infected IN by 10⁴ TCID₅₀ of the indicated rB/HPIV3-RSV G vectors (lanes 1-4) or 10⁶ PFU of wt RSV (lane 5). Sera were collected on day 28 post-immunization. The serum neutralization titers were determined by 60% plaque reduction neutralization assay (PRNT₆₀) on Vero cell monolayers in the presence of added complement. Neutralization titers against wt RSV, with added complement are shown. Lane 1, empty rB/HPIV3 vector; lane 2, wt F, unmodified wild-type RSV F, specifically the “Non-HEK/non-opt” construct that was described in a patent application (PCT/US2016/014154) and a previous publication (Liang, et al., J. Virol. 89: 9499-9510, 2015); lane 3, wt G, wild-type RSV G of A2 strain; lane 4, wt G/GS-opt, a GenScript optimized wild-type G of A2 strain, having the same amino acid sequence as the wt G construct in lane 3; lane 5, wt RSV, wild-type RSV A2 as control. The limit of detection is indicated with a dashed line. Each dot represents the titer of an individual animal. The horizontal lines indicate the mean titers of the groups. The statistical significance of differences in mean titers among all groups in each assay was analyzed using unpaired two-tailed student t-test: * indicates P<0.05; ns=not significant.

FIGS. 12A-12B Replication of wt RSV challenge in hamsters previously infected with the indicated rB/HPIV3-RSV-G or RSV-F vector or wt RSV. Hamsters infected in FIG. 11 were challenged intranasally 30 days later with 10⁶ PFU of wt RSV in total volume of 100 ul in both nostrils. Nasal turbinates (NT) and lungs were collected on day 3 after challenge and homogenized. Titers of RSV in nasal turbinates (FIG. 12A) and lungs (FIG. 12B) were determined by plaque assays in Vero monolayers. The horizontal lines indicate the mean value of titers in that group. Limit of detected is shown by a dashed line.

FIG. 13. Evaluation of the ability of serum RSV-neutralizing antibodies to block RSV-GFP infection in an in vitro model of primary differentiated mucociliary human airway epithelial (HAE) tissue. Aliquots of RSV-GFP were incubated for 30 min at 37° C. with the indicated hamster serum in the absence of added complement, from the experiment shown in FIGS. 7 and 8, and were then inoculated onto HAE cultures (one well per sample). After 60 min at 37° C., the inoculum was removed and the cultures were incubated for 48 hours. The GFP foci of infected cells were imaged and quantified, and the mean of each set of duplicates was calculated. Significant differences were identified by an unpaired t test: ** indicates P<0.01; ns=not significant.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The Sequence Listing is submitted as an ASCII text file in the form of the file name “Sequence.txt’ (˜256 kb), which was created on Nov. 25, 2019, and which is incorporated by reference herein.

DETAILED DESCRIPTION

Major challenges to developing pediatric vaccines against RSV and HPIV3 include the immaturity of the immune system during infancy, immune-suppression by maternal antibodies, inefficient immune protection at the superficial epithelium of the respiratory tract, and vaccine-induced enhanced disease that has been observed in studies with inactivated or subunit RSV and HPIV3 vaccines in virus-naïve recipients (Kim et al., Amer. J. Epidemiol. 89:422-434, 1969; Ottolini et al., Viral Immunol. 13:231-236, 2000; Schneider-Ohrum et al., J. Virol. 91:e02180-16, 2017). Further, although immunization with a live-attenuated rB/HPIV3 vector expressing an RSV antigen (unmodified RSV F protein) did not prime vaccine-induced enhanced disease, clinical trial assessment revealed disappointing RSV immunogenicity (Bernstein, et al. 2012. Pediatric Infectious Disease Journal 31:109-114). Thus, despite substantial effort, a need remains for an effective immunogen that induces a protective immune response to RSV and/or HPIV3.

The present disclosure provides recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3) vectors expressing RSV G or variants thereof (“rB/HPIV3-RSV G” vectors) that meet the above-discussed need. For example, as described in the examples, of nine different rB/HPIV3-RSV G vectors, one vector (rB/HPIV3 comprising a heterologous gene encoding wt RSV G) produced an immune response to RSV in an animal model that provided titers of serum RSV-neutralizing antibodies assayed in the presence of complement that were not significantly different than those induced by wt RSV infection, even though the RSV was administered at a 10-fold higher dose, was not attenuated, and bears both the F and G neutralization antigens.

I. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.

As used herein, the term “comprises” means “includes.” Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:

Adjuvant: A vehicle used to enhance antigenicity. Adjuvants include a suspension of minerals (alum, aluminum hydroxide, or phosphate) on which antigen is adsorbed; or water-in-oil emulsion, for example, in which antigen solution is emulsified in mineral oil (Freund incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and/or causes influx of macrophages). Immunostimulatory oligonucleotides (such as those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules (a “biological adjuvant”), such as costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-α, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, immune stimulating complex (ISCOM) matrix, and toll-like receptor (TLR) agonists, such as TLR-9 agonists, Poly I:C, or PolyICLC. Adjuvants are described, for example, in Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007.

Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intranasal, the composition (such as a composition including a disclosed rB/HPIV3-RSV G vector) is administered by introducing the composition into the nasal passages of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.

Attenuated: A virus that is “attenuated” or has an “attenuated phenotype” refers to a virus that has decreased virulence compared to a reference virus under similar conditions of infection. Attenuation usually is associated with decreased virus replication as compared to replication of a reference wild-type virus under similar conditions of infection, and thus “attenuation” and “restricted replication” often are used synonymously. In some hosts (typically non-natural hosts, including experimental animals), disease is not evident during infection with a reference virus in question, and restriction of virus replication can be used as a surrogate marker for attenuation. In some embodiments, a disclosed rB/HPIV3-RSV G vector that is attenuated exhibits at least about 10-fold or greater decrease, such as at least about 100-fold or greater decrease in virus titer in the upper or lower respiratory tract of a mammal compared to non-attenuated, wild type virus titer in the upper or lower respiratory tract, respectively, of a mammal of the same species under the same conditions of infection. Examples of mammals include, but are not limited to, humans, mice, rabbits, rats, hamsters, such as for example Mesocricetus auratus, and non-human primates, such as for example Ceropithiecus aethiops. An attenuated rB/HPIV3-RSV G vector may display different phenotypes including without limitation altered growth, temperature sensitive growth, host range restricted growth, or plaque size alteration.

Cytoplasmic Tail (CT): A contiguous region of a transmembrane protein that includes a terminus (either N- or C-terminus) of the protein and extends into the cytoplasm of a cell or enveloped virus from the cytoplasmic surface of the cell membrane or viral envelope. In the case of a type I transmembrane protein, the CT includes the C-terminus of the protein. In the case of a type II transmembrane protein, the CT includes the N-terminus of the protein.

Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

Gene: A nucleic acid sequence that comprises control and coding sequences necessary for the transcription of an RNA, whether an mRNA or otherwise. For instance, a gene may comprise a promoter, one or more enhancers or silencers, a nucleic acid sequence that encodes a RNA and/or a polypeptide, downstream regulatory sequences and, possibly, other nucleic acid sequences involved in regulation of the expression of an mRNA.

A “gene” of a rB/HPIV3 vector as described herein refers to a portion of the rB/HPIV3 genome encoding an mRNA and typically begins at the upstream (3′) end with a gene-start (GS) signal and ends at the downstream (5′) end with the gene-end (GE) signal. In this context, the term gene also embraces what is referred to as a “translational open reading frame”, or ORF, particularly in the case where a protein, such as C, is expressed from an additional ORF rather than from a unique mRNA. To construct a disclosed rB/HPIV3 vector, one or more genes or genome segments may be deleted, inserted or substituted in whole or in part.

Heterologous: Originating from a different genetic source. A heterologous gene included in a recombinant genome is a gene that does not originate from that genome. In one specific, non-limiting example, a heterologous gene encoding an ectodomain of a RSV G protein is included in the genome of a rB/HPIV3 vector as described herein.

Host cells: Cells in which a vector can be propagated and its nucleic acid expressed. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.

Infectious and Self-Replicating Virus: A virus that is capable of entering and replicating in a cultured cell or cell of an animal or human host to produce progeny virus capable of the same activity.

Immune response: A response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus. In one embodiment, the response is specific for a particular antigen (an “antigen-specific response”). In one embodiment, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the response is a B cell response, and results in the production of specific antibodies.

Immunogenic composition: A preparation of immunogenic material capable of stimulating an immune response, which in some examples can be administered for the prevention, amelioration, or treatment of infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them. Immunogenic compositions comprise an antigen (such as a virus) that induces a measurable T cell response against the antigen, or induces a measurable B cell response (such as production of antibodies) against the antigen. In one example, an immunogenic composition comprises a disclosed rB/HPIV3-RSV G that induces a measurable CTL response against RSV and HPIV3, or induces a measurable B cell response (such as production of antibodies) against RSV and HPIV3, when administered to a subject. For in vivo use, the immunogenic composition will typically include a recombinant virus in a pharmaceutically acceptable carrier and may also include other agents, such as an adjuvant.

Isolated: An “isolated” biological component has been substantially separated or purified away from other biological components, such as other biological components in which the component naturally occurs, such as other chromosomal and extrachromosomal DNA, RNA, and proteins. Proteins, peptides, nucleic acids, and viruses that have been “isolated” include those purified by standard purification methods. Isolated does not require absolute purity, and can include protein, peptide, nucleic acid, or virus molecules that are at least 50% pure, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% pure.

Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule. Non-limiting examples of peptide linkers include glycine-serine linkers.

Nucleic acid molecule: A polymeric form of nucleotides, which may include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form of either type of nucleotide. The term “nucleic acid molecule” as used herein is synonymous with “polynucleotide.” A nucleic acid molecule is usually at least 10 bases in length, unless otherwise specified. The term includes single- and double-stranded forms of DNA. A nucleic acid molecule may include either or both naturally occurring and modified nucleotides linked together by naturally occurring and/or non-naturally occurring nucleotide linkages.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Parainfluenza virus (PIV): A number of enveloped non-segmented negative-sense single-stranded RNA viruses from family Paramyxoviridae that are descriptively grouped together. This includes all of the members of genus Respirovirus (e.g., HPIV1, HPIV3) and a number of members of genus Rubulavirus (e.g. HPIV2, HPIV4, PIV5). PIVs are made up of two structural modules: (1) an internal ribonucleoprotein core, or nucleocapsid, containing the viral genome, and (2) an outer, roughly spherical lipoprotein envelope. The PIV genome is approximately 15,000 nucleotides in length and encodes at least eight polypeptides. These proteins include the nucleocapsid structural protein (NP, NC, or N depending on the genera), the phosphoprotein (P), the matrix protein (M), the fusion glycoprotein (F), the hemagglutinin-neuraminidase glycoprotein (HN), the large polymerase protein (L), and the C and D proteins. The gene order is 3′-N-P-M-F-HN-L-5′, and each gene encodes a separate protein encoding mRNA, with the P gene containing one or more additional open reading frames (ORFs) encoding accessory proteins.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular embodiments, suitable for administration to a subject the carrier may be sterile, and/or suspended or otherwise contained in a unit dosage form containing one or more measured doses of the composition suitable to induce the desired immune response. It may also be accompanied by medications for its use for treatment purposes. The unit dosage form may be, for example, in a sealed vial that contains sterile contents or a syringe for injection into a subject, or lyophilized for subsequent solubilization and administration or in a solid or controlled release dosage.

Polypeptide: Any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). “Polypeptide” applies to amino acid polymers including naturally occurring amino acid polymers and non-naturally occurring amino acid polymer as well as in which one or more amino acid residue is a non-natural amino acid, for example an artificial chemical mimetic of a corresponding naturally occurring amino acid. A “residue” refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic. A polypeptide has an amino terminal (N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide” is used interchangeably with peptide or protein, and is used herein to refer to a polymer of amino acid residues.

Recombinant: A recombinant nucleic acid molecule or protein is one that has a sequence that is not naturally occurring: for example, includes one or more nucleic acid substitutions, deletions or insertions, and/or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished, for example, by chemical synthesis, targeted mutation of a naturally occurring nucleic acid molecule or protein, or, artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant virus is one that includes a genome that includes a recombinant nucleic acid molecule.

Recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3): A chimeric PIV3 comprising a genome comprising a combination of BPIV3 and HPIV3 genes that together make up the full complement of PIV3 genes in the PIV3 genome (N, P, M, F, HN, and L genes). The disclosed rB/HPIV3 vectors are based on a BPIV3 genome having F and HN genes replaced with the corresponding genes from HPIV3 (one example of which is discussed in Schmidt A C et al., J. Virol. 74:8922-8929, 2000). The structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and genome and anti-genome promoters, are BPIV3 structural and functional genetic elements. The rB/HPIV3 vectors described herein are infectious, self-replicating, and attenuated.

In some embodiments, a heterologous gene encoding RSV G protein or variant thereof is inserted between the N and P genes of the rB/HPIV3 genome to generate a “rB/HPIV3-RSV G” vector. The disclosed rB/HPIV3-RSV G vectors are infectious, self-replicating, and attenuated, and can be used to induce a bivalent immune response to RSV and HPIV3 in a subject.

Respiratory Syncytial Virus (RSV): An enveloped non-segmented negative-sense single-stranded RNA virus of the family Pneumoviridae, genus Orthopneumovirus. The RSV genome is ˜15,000 nucleotides in length and includes 10 genes encoding 11 proteins, including the glycoproteins SH, G and F. The F protein mediates fusion, allowing entry of the virus into the cell cytoplasm and also promoting the formation of syncytia. Two antigenic subgroups of human RSV strains have been described, the A and B subgroups, based primarily on differences in the antigenicity of the G glycoprotein. RSV strains for other species are also known, including bovine RSV. Several animal models of infection by human RSV and closely-related animal counterparts are available, including model organisms infected with human RSV, as well as model organisms infected with species-specific RSV, such as use of bRSV infection in cattle (see, e.g., Bern et al., Am J. Physiol. Lung Cell Mol. Physiol., 301: L148-L156, 2011; and Nam and Kun (Eds.). Respiratory Syncytial Virus: Prevention, Diagnosis and Treatment. Nova Biomedical Nova Science Publisher, 2011; and Cane (Ed.) Respiratory Syncytial Virus. Elsevier Science, 2007.)

RSV G protein: An RSV envelope glycoprotein that is a type II membrane protein and facilitates attachment of RSV to host cell membranes.

The RSV G protein is expressed during RSV infection in two forms. One is the full-length transmembrane form (mG), which is expressed on the cell surface and is packaged into the virus particle. The other form is an N-terminally-truncated, secreted form, sG. The full-length G protein (mG) is a type II protein that has an N-terminal cytoplasmic tail (CT, predicted to comprise amino acids 1-37 in strain A2, see FIG. 1B), a hydrophobic transmembrane domain (TM, comprising approximately amino acids 38-65, see FIG. 1B), and an ectodomain (comprising approximately amino acids 66-298). The sG form is relatively abundant in RSV-infected cell cultures, and is produced by alternative translation initiation at the second AUG codon (M48) in the ORF, whose corresponding position in the protein lies within the TM domain (see FIG. 1B). The N-terminus is then subjected to intracellular proteolytic trimming that creates a new N-terminus at N66 (FIG. 1B).

The ectodomain of RSV G protein comprises two large divergent domains that flank a short central conserved region at amino acids 164-186. The divergent domains have a high content of proline, alanine, threonine, and serine amino acids, and (for strain A2) an estimated four N-linked and 24-25 O-linked carbohydrate side chains. The central conserved domain contains a cysteine noose (i.e., a tight turn stabilized by two disulfide bonds) that bears a conserved CX3C motif (CWAIC, amino acids 182-186 of the A2 strain). The mG and sG forms are believed to be essentially the same with regard to glycosylation and protein structure except that mG forms a multimer that probably is a trimer or tetramer, whereas sG remains a monomer.

An exemplary RSV G protein sequence is provided herein as SEQ ID NO: 22.

Sequence identity: The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs, orthologs, or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

When determining sequence identity between two sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^(th) ed, Cold Spring Harbor, N.Y., 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013).

Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989).

In one examples, once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, a peptide sequence that has 1166 matches when aligned with a test sequence having 1554 amino acids is 75.0 percent identical to the test sequence (1166÷1554*100=75.0). The percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The length value will always be an integer.

Homologs and variants of a polypeptide (such as a RSV G ectodomain) are typically characterized by possession of at least about 75%, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. As used herein, reference to “at least 90% identity” or similar language refers to “at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identity” to a specified reference sequence.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals. In an example, a subject is a human. In a particular example, the subject is a newborn infant. In an additional example, a subject is selected that is in need of inhibiting an RSV infection and/or a HPIV3 infection. For example, the subject is either uninfected and at risk of RSV infection and/or HPIV3 infection or is infected in need of treatment.

Transmembrane domain (TM): An amino acid sequence that spans a lipid bilayer, such as the lipid bilayer of a cell or virus or virus-like particle.

Vaccine: A preparation of immunogenic material capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of infectious or other types of disease. The immunogenic material may include attenuated or killed microorganisms (such as bacteria or viruses), or antigenic proteins, peptides or DNA derived from them. An attenuated vaccine is a virulent organism that has been modified to produce a less virulent form, but nevertheless retains the ability to elicit antibodies and cell-mediated immunity against the virulent form. An inactivated (killed) vaccine is a previously virulent organism that has been inactivated with chemicals, heat, or other treatment, but elicits antibodies against the organism. Vaccines may elicit both prophylactic (preventative or protective) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Vaccines may be administered with an adjuvant to boost the immune response.

Vector: An entity containing a DNA or RNA molecule bearing a promoter(s) that is operationally linked to the coding sequence of an antigen(s) of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication-incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.

II. rB/HPIV3-RSV G Vectors

Recombinant chimeric viral vectors comprising a BPIV3 genome with the encoding sequences of the BPIV3 HN and F genes replaced by encoding sequences of the corresponding HPIV3 HN and F gene, and further comprising a heterologous gene encoding a RSV G protein (such as a wild-type RSV G protein) or a variant thereof are provided herein. These recombinant chimeric viral vectors are referred as “rB/HPIV3-RSV G” vectors.

The rB/HPIV3-RSV G genome contains a full complement of PIV3 genes. Therefore, the rB/HPIV3-RSV G vectors are infectious and replication-competent, but are attenuated in rhesus monkeys and humans due to the BPIV3 backbone, and the presence of the heterologous gene.

The genome of the rB/HPIV3-RSV G vectors comprises the heterologous gene encoding RSV G or a variant thereof, HPIV3 F and HN genes, BPIV3 N, P, M, and L genes, and BPIV3 genomic promoter (3′ leader region) and 5′ trailer region, with the order of 3′-leader region—BPIV3 N, heterologous gene, BPIV3 P, BPIV3 M, HPIV3 F, HPIV3 HN, BPIV3 L—5′-trailer. Exemplary nucleic acid sequences of these genes and proteins encoded thereby are provided herein, as are structural and functional genetic elements that control gene expression, such as gene start and gene end sequences and genome and anti-genome promoters.

An exemplary BPIV3 genome sequence (Kansas stain) is provided as GenBank Acc. No. AF178654.1, which is incorporated by reference herein in its entirety. An exemplary HPIV3 JS strain genome sequence is provided as GenBank Acc. No. Z11575.1, which is incorporated by reference herein in its entirety. In some embodiments, sequences from these strains can be used to construct the rB/HPIV3 aspect of the rB/HPIV3-RSV G vector, for example, as described in Schmidt et al., (J. Virol. 74:8922-8929, 2000). In some such embodiments, the HN protein encoded by the HPIV3 HN gene can be modified to have threonine and proline residues at positions 263 and 370, respectively.

In some embodiments, the rB/HPIV3-RSV G vector comprises a genome comprising HPIV3 F and HN genes and BPIV3 N, P, M, and L genes encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins as set forth below, or encoding HPIV3 F and HN proteins and BPIV3 N, P, C, V, M, and L proteins individually having at least 90% (such as at least 95% or at least 98%) sequence identity to the corresponding HPIV3 F and HN protein or BPIV3 N, P, C, V, M, and L protein set forth below:

BPIV3 N (GenBank Acc. No.: AAF28254.1, encoded by nucleotides 111-1658 of GenBank No. AF178654.1) (SEQ ID NO: 1) MLSLFDTFSARRQENITKSAGGAVIPGQKNTVSIFALGPSITDDNDKMTLALLFLSHSLDNEKQHAQRAGFLVSLLSMAY ANPELYLTSNGSNADVKYVIYMIEKDPGRQKYGGFVVKTREMVYEKTTDWMFGSDLEYDQDNMLQNGRSTSTIEDLVHTF GYPSCLGALIIQVWIILVKAITSISGLRKGFFTRLEAFRQDGTVKSSLVLSGDAVEQIGSIMRSQQSLVTLMVETLITMN TGRNDLTTIEKNIQIVGNYIRDAGLASFFNTIRYGIETRMAALTLSTLRPDINRLKALIELYLSKGPRAPFICILRDPVH GEFAPGNYPALWSYAMGVAVVQNKAMQQYVTGRSYLDIEMFQLGQAVARDAESQMSSILEDELGVTQEAKQSLKKHMKNI SSSDTTFHKPTGGSAIEMAIDEEAGQPESRGDQDQGDEPRSSIVPYAWADETGNDNQTESTTEIDSIKTEQRNIRDRLNK RLNEKRKQSDPRSTDITNNTNQTEIDDLFSAFGSN BPIV3 P (GenBank Acc. No.: AAF28255, encoded by nucleotides 1784-3574 of GenBank No. AF178654) (SEQ ID NO: 2) MEDNVQNNQIMDSWEEGSGDKSSDISSALDIIEFILSTDSQENTADSNEINTGTTRLSTTIYQPESKTTETSKENSGPAN KNRQFGASHERATETKDRNVNQETVQGGYRRGSSPDSRTETMVTRRISRSSPDPNNGTQIQEDIDYNEVGEMDKDSTKRE MRQFKDVPVKVSGSDAIPPTKQDGDGDDGRGLESISTFDSGYTSIVTAATLDDEEELLMKNNRPRKYQSTPQNSDKGIKK GVGRPKDTDKQSSILDYELNFKGSKKSQKILKASTNTGEPTRPQNGSQGKRITSWNILNSESGNRTESTNQTHQTSTSGQ NHTMGPSRTTSEPRIKTQKTDGKEREDTEESTRFTERAITLLQNLGVIQSAAKLDLYQDKRVVCVANVLNNADTASKIDF LAGLMIGVSMDHDTKLNQIQNEILSLKTDLKKMDESHRRLIENQKEQLSLITSLISNLKIMTERGGKKDQPEPSGRTSMI KTKAKEEKIKKVRFDPLMETQGIEKNIPDLYRSIEKTPENDTQIKSEINRLNDESNATRLVPRRISSTMRSLIIIINNSN LSSKAKQSYINELKLCKSDEEVSELMDMFNEDVSSQ BPIV3 C (encoded by nucleotide 1794-2399 of GenBank No. AF178654) (SEQ ID NO: 3) MFKTIKSWILGKRDQEINHLTSHRPSTSLNSYSAPTPKRTRQTAMKSTQEPQDLARQSTNLNPKQQKQARKIVDQLTKID SLGHHTNVPQRQKIEMLIRRLYREDIGEEAAQIVELRLWSLEESPEAAQILTMEPKSRKILITMKLERWIRTLLRGKCDN LKMFQSRYQEVMPFLQQNKMETVMMEEAWNLSVHLIQDIPV BPIV3 V (encoded by nucleotide 1784-3018 of GenBank No. AF178654 with an inserted nucleotide g between nucleotide 2505-2506 at a gene editing site located at nucleotide 2500-2507) (SEQ ID NO: 4) MEDNVQNNQIMDSWEEGSGDKSSDISSALDIIEFILSTDSQENTADSNEINTGTTRLSTTIYQPESKTTETSKENSGPAN KNRQFGASHERATETKDRNVNQETVQGGYRRGSSPDSRTETMVTRRISRSSPDPNNGTQIQEDIDYNEVGEMDKDSTKRE MRQFKDVPVKVSGSDAIPPTKQDGDGDDGRGLESISTFDSGYTSIVTAATLDDEEELLMKNNRPRKYQSTPQNSDKGIKK GGWKAKRHRQTIINIGLRTQLQRIEEEPENPQSQHEYRRTNKTTEWIPGEENHILEHPQQRERQSNRINKPNPSDINLGT EPHNGTKQNNLRTKDQDTKDGWKGKRGHRREHSIYRKGDYIITESWCNPICSKIRPIPRQESCVCGECPKQCRYCIKDRL PSRFDDRSVNGS BPIV3 M (GenBank Acc. No.: AAF28256, encoded by nucleotides 3735-4790 of GenBank No. AF178654) (SEQ ID NO: 5) MSITNSTIYTFPESSFSENGNIEPLPLKVNEQRKAIPHIRVVKIGDPPKHGSRYLDVFLLGFFEMERSKDRYGSISDLDD DPSYKVCGSGSLPLGLARYTGNDQELLQAATKLDIEVRRTVKATEMIVYTVQNIKPELYPWSSRLRKGMLFDANKVALAP QCLPLDRGIKFRVIFVNCTAIGSITLFKIPKSMALLSLPNTISINLQVHIKTGVQTDSKGVVQILDEKGEKSLNFMVHLG LIKRKMGRMYSVEYCKQKIEKMRLLFSLGLVGGISFHVNATGSISKTLASQLAFKREICYPLMDLNPHLNSVIWASSVEI TRVDAVLQPSLPGEFRYYPNIIAKGVGKIRQ HPIV3 F (encoded by nucleotides 5072-6691 of GenBank No. Z11575) (SEQ ID NO: 6) MPTSILLIITTMIMASFCQIDITKLQHVGVLVNSPKGMKISQNFETRYLILSLIPKIEDSNSCGDQQIKQYKKLLDRLII PLYDGLRLQKDVIVTNQESNENTDPRTKRFFGGVIGTIALGVATSAQITAAVALVEAKQARSDIEKLKEAIRDTNKAVQS VQSSIGNLIVAIKSVQDYVNKEIVPSIARLGCEAAGLQLGIALTQHYSELTNIFGDNIGSLQEKGIKLQGIASLYRTNIT EIFTTSTVDKYDIYDLLFTESIKVRVIDVDLNDYSITLQVRLPLLTRLLNTQIYKVDSISYNIQNREWYIPLPSHIMTKG AFLGGADVKECIEAFSSYICPSDPGFVLNHEIESCLSGNISQCPRTTVTSDIVPRYAFVNGGVVANCITTTCTCNGIGNR INQPPDQGVKIITHKECSTIGINGMLFNTNKEGTLAFYTPNDITLNNSVALDPIDISIELNKAKSDLEESKEWIRRSNQK LDSIGNWHQSSTTIIIILIMIIILFIINITIITIAIKYYRIQKRNRVDQNDKPYVLTNK HPIV3 wt HN (encoded by nucleotides 6806-8524 of GenBank No. Z11575) (SEQ ID NO: 7) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFIIVLINSIKSEKAHESLLQDINNEFMEITEKI QMASDNTNDLIQSGVNTRLLTIQSHVQNYIPISLTQQMSDLRKFISEITIRNDNQEVLPQRITHDVGIKPLNPDDFWRCT SGLPSLMKTPKIRLMPGPGLLAMPTTVDGCVRTPSLVINDLIYAYTSNLITRGCQDIGKSYQVLQIGIITVNSDLVPDLN PRISHTFNINDNRKSCSLALLNTDVYQLCSTPKVDERSDYASSGIEDIVLDIVNYDGSISTTRFKNNNISFDQPYAALYP SVGPGIYYKGKIIFLGYGGLEHPINENVICNTTGCPGKTQRDCNQASHSPWFSDRRMVNSIIVVDKGLNSIPKLKVWTIS MRQNYWGSEGRLLLLGNKIYIYTRSTSWHSKLQLGIIDITDYSDIRIKWTWHNVLSRPGNNECPWGHSCPDGCITGVYTD AYPLNPTGSIVSSVILDSQKSRVNPVITYSTATERVNELAILNRTLSAGYTTTSCITHYNKGYCFHIVEINHKSLNTFQP MLFKTEIPKSCS

In some embodiments, the HPIV3 HN gene in rB/HPIV3 vector encodes a HPIV3 HN protein comprising the amino acid sequence set forth as:

(SEQ ID NO: 8) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFI IVLINSIKSEKAHESLLQDINNEFMEITEKIQMASDNTNDLIQSGVNTR LLTIQSHVQNYIPISLTQQMSDLRKFISEITIRNDNQEVLPQRITHDVG IKPLNPDDFWRCTSGLPSLMKTPKIRLMPGPGLLAMPTTVDGCVRTPSL VINDLIYAYTSNLITRGCQDIGKSYQVLQIGIITVNSDLVPDLNPRISH TFNINDNRKSCSLALLNIDVYQLCSTPKVDERSDYASSGIEDIVLDIVN YDGSISTTRFKNNNISFDQPYAALYPSVGPGIYYKGKIIFLGYGGLEHP INENVICNTTGCPGKTQRDCNQASHSTWFSDRRMVNSIIVVDKGLNSIP KLKVWTISMRQNYWGSEGRLLLLGNKIYIYTRSTSWHSKLQLGIIDITD YSDIRIKWTWHNVLSRPGNNECPWGHSCPDGCITGVYTDAYPLNPTGSI VSSVILDSQKSRVNPVITYSTATERVNELAILNRTLSAGYTTTSCITHY NKGYCFHIVEINHKSLNTFQPMLFKTEIPKSCS

The HN protein shown as SEQ ID NO: 7 comprises 263T and 370P amino acid assignments. As discussed in the examples, rB/HPIV3-RSV G including an HN protein with 263T and 370P amino acid assignments can be recovered and passaged with substantially reduced occurrence of adventitious mutations, which increases the efficiency of virus production, analysis, and manufacture. Any of the rB/HPIV3-RSV G vectors provided herein can comprise a HPIV3 HN gene encoding HN protein with 263T and 370P amino acid assignments (for example, introduced into the HN protein by I263T and T370P amino acid substitutions). An exemplary DNA sequence encoding SEQ ID NO: 7 is provided as follows:

(SEQ ID NO: 9) atggaatactggaagcataccaatcacggaaaggatgctggtaatgagc tggagacgtctatggctactcatggcaacaagctcactaataagataat atacatattatggacaataatcctggtgttattatcaatagtcttcatc atagtgctaattaattccatcaaaagtgaaaaggcccacgaatcattgc tgcaagacataaataatgagtttatggaaattacagaaaagatccaaat ggcatcggataataccaatgatctaatacagtcaggagtgaatacaagg cttcttacaattcagagtcatgtccagaattacataccaatatcattga cacaacagatgtcagatcttaggaaattcattagtgaaattacaattag aaatgataatcaagaagtgctgccacaaagaataacacatgatgtaggt ataaaacctttaaatccagatgatttttggagatgcacgtctggtcttc catctttaatgaaaactccaaaaataaggttaatgccagggccgggatt attagctatgccaacgactgttgatggctgtgttagaactccgtcttta gttataaatgatctgatttatgcttatacctcaaatctaattactcgag gttgtcaggatataggaaaatcatatcaagtcttacagatagggataat aactgtaaactcagacttggtacctgacttaaatcctaggatctctcat acctttaacataaatgacaataggaagtcatgttctctagcactcctaa atatagatgtatatcaactgtgttcaactcccaaagttgatgaaagatc agattatgcatcatcaggcatagaagatattgtacttgatattgtcaat tatgatggttcaatctcaacaacaagatttaagaataataacataagct ttgatcaaccatatgctgcactatacccatctgttggaccagggatata ctacaaaggcaaaataatatttctcgggtatggaggtcttgaacatcca ataaatgagaatgtaatctgcaacacaactgggtgccccgggaaaacac agagagactgtaatcaagcatctcatagtacttggttttcagataggag gatggtcaactccatcattgttgttgacaaaggcttaaactcaattcca aaattgaaagtatggacgatatctatgcgacaaaattactgggggtcag aaggaaggttacttctactaggtaacaagatctatatatatacaagatc tacaagttggcatagcaagttacaattaggaataattgatattactgat tacagtgatataaggataaaatggacatggcataatgtgctatcaagac caggaaacaatgaatgtccatggggacattcatgtccagatggatgtat aacaggagtatatactgatgcatatccactcaatcccacagggagcatt gtgtcatctgtcatattagactcacaaaaatcgagagtgaacccagtca taacttactcaacagcaaccgaaagagtaaacgagctggccatcctaaa cagaacactctcagctggatatacaacaacaagctgcattacacactat aacaaaggatattgttttcatatagtagaaataaatcataaaagcttaa acacatttcaacccatgttgttcaaaacagagattccaaaaagctgcag ttaa  BPIV3 L (GenBank Acc. No.: AAF28259, encoded by nucleotides 8640-15341 of GenBank No. AF178654) (SEQ ID NO: 10) MDTESHSGTTSDILYPECHLNSPIVKGKIAQLHTIMSLPQPYDMDDDSI LIITRQKIKLNKLDKRQRSIRKLRSVLMERVSDLGKYTFIRYPEMSSEM FQLCIPGINNKINELLSKASKTYNQMTDGLRDLWVTILSKLASKNDGSN YDINEDISNISNVHMTYQSDKWYNPFKTWFTIKYDMRRLQKAKNEITFN RHKDYNLLEDQKNILLIHPELVLILDKQNYNGYIMTPELVLMYCDVVEG RWNISSCAKLDPKLQSMYYKGNNLWEIIDGLFSTLGERTFDIISLLEPL ALSLIQTYDPVKQLRGAFLNHVLSEMELIFAAECTTEEIPNVDYIDKIL DVFKESTIDEIAEIFSFFRTFGHPPLEASIAAEKVRKYMYTEKCLKFDT INKCHAIFCTIIINGYRERHGGQWPPVTLPVHAHEFIINAYGSNSAISY ENAVDYYKSFIGIKFDKFIEPQLDEDLTIYMKDKALSPKKSNWDTVYPA SNLLYRTNVSHDSRRLVEVFIADSKFDPHQVLDYVESGYWLDDPEFNIS YSLKEKEIKQEGRLFAKMTYKMRATQVLSETLLANNIGKFFQENGMVKG EIELLKRLTTISMSGVPRYNEVYNNSKSHTEELQAYNAISSSNLSSNQK SKKFEFKSTDIYNDGYETVSCFLTTDLKKYCLNWRYESTALFGDTCNQI FGLKELFNWLHPRLEKSTIYVGDPYCPPSDIEHLPLDDHPDSGFYVHNP KGGIEGFCQKLWTLISISAIHLAAVKIGVRVTAMVQGDNQAIAVTTRVP NNYDYKVKKEIVYKDVVRFFDSLREVMDDLGHELKLNETIISSKMFIYS KRIYYDGRILPQALKALSRCVFWSETIIDETRSASSNLATSFAKAIENG YSPVLGYVCSIFKNIQQLYIALGMNINPTITQNIKDQYFRNIHWMQYAS LIPASVGGFNYMAMSRCFVRNIGDPTVAALADIKRFIKANLLDRGVLYR IMNQEPGESSFLDWASDPYSCNLPQSQNITTMIKNITARNVLQDSPNPL LSGLFTSTMIEEDEELAEFLMDRRIILPRVAHDILDNSLTGIRNAIAGM LDTTKSLIRVGISRGGLTYNLLRKISNYDLVQYETLSKTLRLIVSDKIK YEDMCSVDLAISLRQKMWMHLSGGRMINGLETPDPLELLSGVIITGSEH CRICYSTEGESPYTWMYLPGNLNIGSAETGIASLRVPYFGSVTDERSEA QLGYIKNLSKPAKAAIRIAMIYTWAFGNDEISWMEASQIAQTRANFTLD SLKILTPVTTSTNLSHRLKDTATQMKFSSTSLIRVSRFITISNDNMSIK EANETKDTNLIYQQVMLTGLSVFEYLFRLEESTGHNPMVMHLHIEDGCC IKESYNDEHINPESTLELIKYPESNEFIYDKDPLKDIDLSKLMVIRDHS YTIDMNYWDDTDIVHAISICTAVTIADTMSQLDRDNLKELVVIANDDDI NSLITEFLTLDILVFLKTFGGLLVNQFAYTLYGLKIEGRDPIWDYIMRT LKDTSHSVLKVLSNALSHPKVFKRFWDCGVLNPIYGPNTASQDQVKLAL SICEYSLDLFMREWLNGASLEIYICDSDMEIANDRRQAFLSRHLAFVCC LAEIASFGPNLLNLTYLERLDELKQYLDLNIKEDPTLKYVQVSGLLIKS FPSTVTYVRKTAIKYLRIRGINPPETIEDWDPIEDENILDNIVKTVNDN CSDNQKRNKSSYFWGLALKNYQVVKIRSITSDSEVNEASNVTTHGMTLP QGGSYLSHQLRLFGVNSTSCLKALELSQILMREVKKDKDRLFLGEGAGA MLACYDATLGPAINYYNSGLNITDVIGQRELKIFPSEVSLVGKKLGNVT QILNRVRVLFNGNPNSTWIGNMECESLIWSELNDKSIGLVHCDMEGAIG KSEETVLHEHYSIIRITYLIGDDDVVLVSKIIPTITPNWSKILYLYKLY WKDVSVVSLKTSNPASTELYLISKDAYCTVMEPSNLVLSKLKRISSIEE NNLLKWIILSKRKNNEWLQHEIKEGERDYGIMRPYHTALQIFGFQINLN HLAREFLSTPDLTNINNIIQSFTRTIKDVMFEWVNITHDNKRHKLGGRY NLFPLKNKGKLRLLSRRLVLSWISLSLSTRLLTGRFPDEKFENRAQTGY VSLADIDLESLKLLSRNIVKNYKEHIGLISYWFLTKEVKILMKLIGGVK LLGIPKQYKELEDRSSQGYEYDNEFDID

The encoding sequences of the HPIV3 F and HN genes and the BPIV3 N, P, M, and L genes in the rB/HPIV3-RSV G vector are flanked by appropriate gene start and gene-end sequences to facilitate expression from the viral genome. For example, in some embodiments, the encoding sequences of the HPIV3 F and HN genes and the BPIV3 N, P, M, and L genes can be flanked by BPIV3 gene-start and gene end sequences as follows:

SEQ SEQ Gene Gene start ID Gene end ID N aggattaaagac 11 aaataagaaaaa 16 P aggattaaag 12 aaataagaaaaa 17 M aggattaaag 12 aaataaaggataatcaaaaa 18 F aggacaaaag 13 aattataaaaaa 19 HN aggagtaaag 14 aaatataaaaaa 20 L aggagcaaag 15 aaagtaagaaaaa 21

Further, the rB/HPIV3-RSV G vector comprises appropriate genome and anti-genome promoters, such as those of the BPIV3 Kansas strain as set forth in GenBank Acc. No. AF178654, which provides genomic promoter as nucleotides 1-96 and the antigenomic promoter as nucleotides 15361-15456.

The genome of the rB/HPIV3-RSV G comprises a heterologous gene encoding a native RSV G protein or a variant thereof, such as a recombinant RSV G protein comprising BPIV3 HN transmembrane and/or cytoplasmic tail sequences in place of the native RSV G transmembrane and/or cytoplasmic tail sequences. Human RSV can be classified into two groups: A and B. Groups A and B include subgroups A1, A2, B1, and B2, based mainly on sequence variability of the attachment (G) and fusion (F) proteins. The heterologous gene included in the genome of the rB/HPIV3-RSV G can encode a RSV G protein and/or an RSV G ectodomain from (or derived from) any human RSV group (such as Group A or Group B) or subgroup of human RSV (such as subgroup A1, A2, B1, or B2).

An exemplary human RSV G protein sequence from subgroup A2 is set forth below:

(SEQ ID NO: 22) MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAMI ISTSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLG ISPSNPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPT TKQRQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGK KTTTKPTKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIIT TLLTSNTTGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPN TPRQ

In some embodiments, the heterologous gene included in the genome of the rB/HPIV3-RSV G encodes a RSV G protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 22, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 22

An exemplary human RSV G ectodomain sequence from subgroup A2 is set forth below:

(SEQ ID NO: 23) NHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEITSQITTI LASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPPSKPNNDF HFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTLKTTK KDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTGNPELTSQ METFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In some embodiments, the heterologous gene encodes a recombinant RSV G ectodomain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 23, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 23. The ectodomain is linked to appropriate transmembrane domain and cytoplasmic tail sequences, such as those set forth herein.

An exemplary human RSV G protein sequence from RSV A/Maryland/001/11 is set forth below:

(SEQ ID NO: 47) MSKTKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSIAQITLSILAMI ISTSLIIAAIIFIASANHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLG ISLSNLSETTSKPTTILALTTPNAESTPQSTTVKTKNTTTTQIQPSKPT TKQRQNKPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGR KTTTKPTKQPAIKTTKKDPKPQTTKPKEVLTTKPTEKPTINTTKTNIRT TLLTSNITENQEHTSQKETLHSTTSEGNPSPSQVYTTSEYLSQSLSPSN TTRW

In some embodiments, the heterologous gene included in the genome of the rB/HPIV3-RSV G encodes a RSV G protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 47, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 47.

An exemplary human RSV G ectodomain sequence from RSV A/Maryland/001/11 is set forth below:

(SEQ ID NO: 48) NHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISLSNLSETTSKPTTI LALTTPNAESTPQSTTVKTKNTTTTQIQPSKPTTKQRQNKPQNKPNNDF HFEVFNFVPCSICSNNPTCWAICKRIPNKKPGRKTTTKPTKQPAIKTTK KDPKPQTTKPKEVLTTKPTEKPTINTTKTNIRTTLLTSNITENQEHTSQ KETLHSTTSEGNPSPSQVYTTSEYLSQSLSPSNTTRW

In some embodiments, the heterologous gene encodes a recombinant RSV G ectodomain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 48, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 48. The ectodomain is linked to appropriate transmembrane domain and cytoplasmic tail sequences, such as those set forth herein.

An exemplary human RSV G protein sequence from Subgroup B (B1, AAB82435.1, see Karron et al., Proc. Natl. Acad. Sci. U.S.A. 94, 13961-6, 1997) is set forth below:

(SEQ ID NO: 49) MSKHKNQRTARTLEKTWDTLNHLIVISSCLYRLNLKSIAQIALSVLAMI ISTSLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVPPER VSSSKQPTTTSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPS TKPRLKNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKK KPTIKPTNKPTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTS TSQSTVLDTTTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQN TQSHA

In some embodiments, the heterologous gene included in the genome of the rB/HPIV3-RSV G encodes a RSV G protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 49, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 49.

An exemplary human RSV G ectodomain sequence from Subgroup B (B1, AAB82435.1) is set forth below:

(SEQ ID NO: 50) NHKVILTIVIVQTIKNHTEKNITTYLTQVPPERVSSSKQPITTSPIHTN SATTSPNIKSETHHTTAQTKGRITTSTQINKPSTKPRLKNPPKKPKDDY HFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPINKPTIKTIN KRDPKTPAKTIKKETTINPIKKPTLITTERDTSTSQSTVLDTTTLEHTI QQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA

In some embodiments, the heterologous gene encodes a recombinant RSV G ectodomain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 50, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 50. The ectodomain is linked to appropriate transmembrane domain and cytoplasmic tail sequences, such as those set forth herein.

An exemplary human RSV G protein sequence from Subgroup A (genotype ON1; ON67-1210A, AEQ98758.1, Eshagi A. et al., Plos One 7(3):e32807, 2012 is set forth below:

(SEQ ID NO: 51) MSKTKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSIAQITLSILAMI ISTSLIIAAIIFIASANHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLG ISFSNLSGTTSQSTTILASTTPSAESTPQSTTVKIKNTTTTQILPSKPT TKQRQNKPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGK KTTTKPTKKPTLKTTKKDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRT TLLTSNTKGNPEHTSQEETLHSTTSEGYLSPSQVYTTSGQEETLHSTTS EGYLSPSQVYTTSEYLSQSLSSSNTTK

In some embodiments, the heterologous gene included in the genome of the rB/HPIV3-RSV G encodes a RSV G protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 51, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 51.

An exemplary human RSV G ectodomain sequence from Subgroup A (genotype ON1; ON67-1210A, AEQ98758.1, Eshagi A. et al., Plos One 7(3):e32807, 2012) is set forth below:

(SEQ ID NO: 52) NHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISFSNLSGTTSQSTTI LASTTPSAESTPQSTTVKIKNTTTTQILPSKPTTKQRQNKPQNKPNNDF HFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKKPTLKTTK KDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRTTLLTSNTKGNPEHTSQ EETLHSTTSEGYLSPSQVYTTSGQEETLHSTTSEGYLSPSQVYTTSEYL SQSLSSSNTTK

In some embodiments, the heterologous gene encodes a recombinant RSV G ectodomain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 52, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 52. The ectodomain is linked to appropriate transmembrane domain and cytoplasmic tail sequences, such as those set forth herein.

An exemplary human RSV G protein sequence from Subgroup B (genotype BA1; BA4128/99B, AAQ16179.1, Trento A. et al., J. Gen. Virol. 84, 3115-3120, 2003) is set forth below:

(SEQ ID NO: 53) MSKNKNQRTARTLEKTWDTLNHLIVISSCLYKLNLKSIAQIALSVLAMI ISTSLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVSPER VSPSKQLTTTPPIYTNSATISPNTKSETHHTTAQTKGRTTTPTQNNKPS TKPRPKNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKK KPTIKPTNKPPTKTTNKRDPKKLAKTLKKETTINPTKKPTPKTTERDTS TSQSTVLDTTTSKHTERDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENT PNSTQTPTASEPSTSNSTQKL

In some embodiments, the heterologous gene included in the genome of the rB/HPIV3-RSV G encodes a RSV G protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 53, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 53.

An exemplary human RSV G ectodomain sequence from Subgroup B (genotype BA1; BA4128/99B, AAQ16179.1, Trento A. et al., J. Gen. Virol. 84, 3115-20, 2003) is set forth below:

(SEQ ID NO: 54) NHKVTLTTVTVQTIKNHTEKNITTYLTQVSPERVSPSKQLTTTPPIYTN SATISPNTKSETHHTTAQTKGRTTTPTQNNKPSTKPRPKNPPKKPKDDY HFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNKPPTKTTN KRDPKKLAKTLKKETTINPTKKPTPKTTERDTSTSQSTVLDTTTSKHTE RDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENTPNSTQTPTASEPSTSN STQKL

In some embodiments, the heterologous gene encodes a recombinant RSV G ectodomain comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 54, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 54. The ectodomain is linked to appropriate transmembrane domain and cytoplasmic tail sequences, such as those set forth herein.

In some embodiments, the heterologous gene encodes a recombinant RSV G protein comprising an RSV G ectodomain and transmembrane domain linked to a cytoplasmic tail of a BPIV3 HN protein, a HPIV3 HN protein, or a HPIV1 HN protein. In some embodiments, the heterologous gene encodes a recombinant RSV G protein comprising an RSV G ectodomain linked to a transmembrane domain and cytoplasmic tail of a BPIV3 HN protein, a HPIV3 HN protein, or a HPIV1 HN protein. As discussed in the Examples, swapping the PIV HN transmembrane and cytoplasmic tail protein sequences with the RSV G transmembrane and cytoplasmic tail protein sequences promotes membrane insertion and packaging of the type-II membrane protein into the virion envelope. It is believed that an increase in the amount of RSV G ectodomain exposed on the surface of the virion envelope leads to a corresponding increase in the immune response to the RSV G ectodomain.

The transmembrane domain and cytoplasmic tail of an exemplary RSV G sequence from RSV A2 are set forth as follows:

RSV G CT: SEQ ID NO: 24 MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKS, RSV G TM: SEQ ID NO: 25 VAQITLSILAMIISTSLIIAAIIFIASA. RSV G TM + CT: SEQ ID NO: 26 MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAMI ISTSLIIAAIIFIASA,

Further, the cytoplasmic tail and transmembrane domain of an exemplary BPIV3 HN protein are set forth as follows:

BPIV3 HN CT: SEQ ID NO: 27 MEYWKHTNSINNTNNETETARGKHSSKVTN, BPIV3 HN TM: SEQ ID NO: 28 IIMYTFWTITLTILSVIFIMILTNLI, BPIV3 HN TM + CT: SEQ ID NO: 29 MEYWKHTNSINNTNNETETARGKHSSKVTNIIMYTFWTITLTILSVIFIM ILTNLI,

Further, the cytoplasmic tail and transmembrane domain of an exemplary HPIV3 HN protein are set forth as follows:

HPIV3 HN CT: SEQ ID NO: 55 MEYWKHTNHGKDAGNELETSMATHGNKLTNK, HPIV3 HN TM: SEQ ID NO: 56 IIYILWTIILVLLSIVFIIVLINSI, HPIV3 HN TM + CT: SEQ ID NO: 57 MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFII VLINSI,

Further, the cytoplasmic tail and transmembrane domain of an exemplary HPIV1 HN protein are set forth as follows:

HPIV1 HN CT: SEQ ID NO: 58 MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHW, HPIV1 HN TM: SEQ ID NO: 59 ILLIATTMHTVLSFIIMILCIDLII, HPIV1 HN TM + CT: SEQ ID NO: 60 MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWILLIATTMHTVLSFI IMILCIDLII,

The human RSV G, BPIV3 HN, HPIV3 HN, and HPIV1 HN proteins exhibit remarkable sequence conservation across corresponding viral subgroups. Accordingly, the cytoplasmic tail and transmembrane domain sequences of an RSV G protein can readily be identified and swapped for the corresponding sequences of a BPIV3, HPIV3, or HPIV1 HN protein as needed when constructing the heterologous gene included in the rB/HPIV3-RSV G vector.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV A2, and a BPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 30) MEYWKHTNSINNTNNETETARGKHSSKVTNVAQITLSILAMIISTSLII AAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPS EITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNK PPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPT KKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNT TGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 30, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 30.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV A2 and BPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 31) MEYWKHTNSINNTNNETETARGKHSSKVTNIIMYTFWTITLTILSVIFI MILTNLINHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEI TSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPP SKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK PTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTG NPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 31, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 31.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV B (B1, GenBank AAB82435.1), and a BPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 61) MEYWKHTNSINNTNNETETARGKHSSKVTNIAQIALSVLAMIISTSLII AAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSSKQP TTTSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRLKN PPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPT NKPTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTVL DTTTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 61, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 61.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV B (B1, GenBank AAB82435.1) and BPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 62) MEYWKHTNSINNTNNETETARGKHSSKVTNIIMYTFWTITLTILSVIFI MILTNLINHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSSKQPTT TSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRLKNPP KKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNK PTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTVLDT TTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 62, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 62.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV A/Maryland/001/11, and a BPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 63) MEYWKHENSINNTNNETETARGKHSSKVENIAQIELSILAMIISTSLII AAIIFIASANHKVELTTAIIQDATNQIKNETPTYLTQNPQLGISLSNLS ETTSKPTTILALTTPNAESTPQSTTVKTKNETTTQIQPSKPETKQRQNK PQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGRKETTKPT KQPAIKTEKKDPKPQTTKPKEVLETKPTEKPTINTEKTNIRTTLLTSNI TENQEHTSQKETLHSTESEGNPSPSQVYTTSEYLSQSLSPSNETRW

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 63, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 63.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV A/Maryland/001/11 and BPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 64) MEYWKHTNSINNTNNETETARGKHSSKVTNIIMYTFWTITLTILSVIFI MILTNLINHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISLSNLSET TSKPTTILALTTPNAESTPQSTTVKTKNTTTTQIQPSKPTTKQRQNKPQ NKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGRKTTTKPTKQ PAIKTTKKDPKPQTTKPKEVLTTKPTEKPTINTTKTNIRTTLLTSNITE NQEHTSQKETLHSTTSEGNPSPSQVYTTSEYLSQSLSPSNTTRW

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 64, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 64.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV ON1, and a BPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 65) MEYWKHTNSINNTNNETETARGKHSSKVTNIAQITLSILAMIISTSLII AAIIFIASANHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISFSNLS GTTSQSTTILASTTPSAESTPQSTTVKIKNTTTTQILPSKPTTKQRQNK PQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPT KKPTLKTTKKDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRTTLLTSNT KGNPEHTSQEETLHSTTSEGYLSPSQVYTTSGQEETLHSTTSEGYLSPS QVYTTSEYLSQSLSSSNTTK

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 65, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 65.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV ON1, and BPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 66) MEYWKHTNSINNTNNETETARGKHSSKVTNIIMYTFWTITLTILSVIFI MILTNLINHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISFSNLSGT TSQSTTILASTTPSAESTPQSTTVKIKNTTTTQILPSKPTTKQRQNKPQ NKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK PTLKTTKKDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRTTLLTSNTKG NPEHTSQEETLHSTTSEGYLSPSQVYTTSGQEETLHSTTSEGYLSPSQV YTTSEYLSQSLSSSNTTK

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 66, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 66.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV BA1, and a BPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 67) MEYWKHTNSINNTNNETETARGKHSSKVTNIAQIALSVLAMIISTSLII AAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVSPERVSPSKQL TTTPPIYTNSATISPNTKSETHHTTAQTKGRTTTPTQNNKPSTKPRPKN PPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPT NKPPTKTTNKRDPKKLAKTLKKETTINPTKKPTPKTTERDTSTSQSTVL DTTTSKHTERDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENTPNSTQTP TASEPSTSNSTQKL

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 67, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 67.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV BA1, and BPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 68) MEYWKHTNSINNTNNETETARGKHSSKVTNIIMYTFWTITLTILSVIFI MILTNLINHKVTLTTVTVQTIKNHTEKNITTYLTQVSPERVSPSKQLTT TPPIYTNSATISPNTKSETHHTTAQTKGRTTTPTQNNKPSTKPRPKNPP KKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNK PPTKTTNKRDPKKLAKTLKKETTINPTKKPTPKTTERDTSTSQSTVLDT TTSKHTERDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENTPNSTQTPTA SEPSTSNSTQKL

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 68, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 68.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV A2, and a HPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 69) MEYWKHTNHGKDAGNELETSMATHGNKLTNKVAQITLSILAMIISTSLI IAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNP SEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQN KPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKP TKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSN TTGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 69, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 69.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV A2 and HPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 70) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFI IVLINSINHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPSEI TSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKPP SKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK PTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTG NPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 70, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 70.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV B (B1, GenBank AAB82435.1), and a HPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 71) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIAQIALSVLAMIISTSLI IAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSSKQ PTTTSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRLK NPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKP TNKPTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTV LDTTTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 71, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 71.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV B (B1, GenBank AAB82435.1) and HPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 72) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFI IVLINSINHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSSKQPTT TSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRLKNPP KKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNK PTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTVLDT TTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 72, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 72.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV A/Maryland/001/11, and a HPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 73) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIAQITLSILAMIISTSLI IAAIIFIASANHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISLSNL SETTSKPTTILALTTPNAESTPQSTTVKTKNTTTTQIQPSKPTTKQRQN KPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGRKTTTKP TKQPAIKTTKKDPKPQTTKPKEVLTTKPTEKPTINTTKTNIRTTLLTSN ITENQEHTSQKETLHSTTSEGNPSPSQVYTTSEYLSQSLSPSNTTRW

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 73, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 73.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV A/Maryland/001/11 and HPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 74) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFI IVLINSINHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISLSNLSET TSKPTTILALTTPNAESTPQSTTVKTKNTTTTQIQPSKPTTKQRQNKPQ NKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGRKTTTKPTKQ PAIKTTKKDPKPQTTKPKEVLTTKPTEKPTINTTKTNIRTTLLTSNITE NQEHTSQKETLHSTTSEGNPSPSQVYTTSEYLSQSLSPSNTTRW

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 74, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 74.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV ON1, and a HPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 75) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIAQITLSILAMIISTSLI IAAIIFIASANHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISFSNL SGTTSQSTTILASTTPSAESTPQSTTVKIKNTTTTQILPSKPTTKQRQN KPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKP TKKPTLKTTKKDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRTTLLTSN TKGNPEHTSQEETLHSTTSEGYLSPSQVYTTSGQEETLHSTTSEGYLSP SQVYTTSEYLSQSLSSSNTTK

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 75, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 75.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV ON1, and HPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 76) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFI IVLINSINHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISFSNLSGT TSQSTTILASTTPSAESTPQSTTVKIKNTTTTQILPSKPTTKQRQNKPQ NKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK PTLKTTKKDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRTTLLTSNTKG NPEHTSQEETLHSTTSEGYLSPSQVYTTSGQEETLHSTTSEGYLSPSQV YTTSEYLSQSLSSSNTTK

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 76, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 76.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV BA1, and a HPIV3 HN cytoplasmic tail is provided below:

(SEQ ID NO: 77) MEYWKHTNHGKDAGNELETSMATHGNKLTNKIAQIALSVLAMIISTSLI IAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVSPERVSPSKQ LTTTPPIYTNSATISPNTKSETHHTTAQTKGRTTTPTQNNKPSTKPRPK NPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKP TNKPPTKTTNKRDPKKLAKTLKKETTINPTKKPTPKTTERDTSTSQSTV LDTTTSKHTERDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENTPNSTQT PTASEPSTSNSTQKL

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 77, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 77.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV BA1, and HPIV3 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 78 MEYWKHTNHGKDAGNELETSMATHGNKLTNKIIYILWTIILVLLSIVFII VLINSINHKVTLTTVTVQTIKNHTEKNITTYLTQVSPERVSPSKQLTTTP PIYTNSATISPNTKSETHHTTAQTKGRTTTPTQNNKPSTKPRPKNPPKKP KDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNKPPTK TTNKRDPKKLAKTLKKETTINPTKKPTPKTTERDTSTSQSTVLDTTTSKH TERDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENTPNSTQTPTASEPSTS NSTQKL

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 78, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 78.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV A2, and a HPIV1 HN cytoplasmic tail is provided below:

(SEQ ID NO: 79) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWVAQITLSILAMIIST SLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPS NPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQ NKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKP TKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNT TGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 79, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 79.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV A2 and HPIV1 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 80) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWILLIATTMHTVLSFI IMILCIDLIINHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGISPSNPS EITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQRQNKP PSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK PTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTSNTTGN PELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 80, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 80.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV B (B1, GenBank AAB82435.1), and a HPIV1 HN cytoplasmic tail is provided below:

(SEQ ID NO: 81) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWIAQIALSVLAMIIST SLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSS KQPTTTSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRL KNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKP TNKPTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTVL DTTTLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 81, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 81.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV B (B1, GenBank AAB82435.1) and HPIV1 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 82) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWILLIATTMHTVLSFI IMILCIDLIINHKVTLTTVTVQTIKNHTEKNITTYLTQVPPERVSSSKQP TTTSPIHTNSATTSPNTKSETHHTTAQTKGRTTTSTQTNKPSTKPRLKNP PKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPTNK PTTKTTNKRDPKTPAKTTKKETTTNPTKKPTLTTTERDTSTSQSTVLDTT TLEHTIQQQSLHSTTPENTPNSTQTPTASEPSTSNSTQNTQSHA

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 82, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 82.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV A/Maryland/001/11, and a HPIV1 HN cytoplasmic tail is provided below:

(SEQ ID NO: 83) MAEKGKINSSYWSTTRNDNSTVNTHINTPAGRTHWIAQITLSILAMIIST SLIIAAIIFIASANHKVILTTAIIQDATNQIKNTTPTYLTQNPQLGISLS NLSETTSKPITILALTTPNAESTPQSTIVKTKNITTTQIQPSKPITKQRQ NKPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGRKTITKP TKQPAIKTTKKDPKPQTTKPKEVLITKPTEKPTINTTKINIRTILLTSNI TENQEHTSQKETLHSTTSEGNPSPSQVYTTSEYLSQSLSPSNTTRW

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 83, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 83.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV A/Maryland/001/11 and HPIV1 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 84) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWILLIATTMHTVLSFI IMILCIDLIINHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISLSNLS ETTSKPTTILALTTPNAESTPQSTTVKTKNTTTTQIQPSKPTTKQRQNKP QNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGRKTTTKPTKQ PAIKTTKKDPKPQTTKPKEVLTTKPTEKPTINTTKTNIRTTLLTSNITEN QEHTSQKETLHSTTSEGNPSPSQVYTTSEYLSQSLSPSNTTRW

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 84, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 84.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV ON1, and a HPIV1 HN cytoplasmic tail is provided below:

(SEQ ID NO: 85) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWIAQITLSILAMIIST SLIIAAIIFIASANHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISFS NLSGTTSQSTTILASTTPSAESTPQSTTVKIKNTTTTQILPSKPTTKQRQ NKPQNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKP TKKPTLKTTKKDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRTTLLTSNT KGNPEHTSQEETLHSTTSEGYLSPSQVYTTSGQEETLHSTTSEGYLSPSQ VYTTSEYLSQSLSSSNTTK

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 85, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 85.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV ON1, and HPIV1 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 86) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWILLIATTMHTVLSFI IMILCIDLIINHKVTLTTAIIQDATNQIKNTTPTYLTQNPQLGISFSNLS GTTSQSTTILASTTPSAESTPQSTTVKIKNTTTTQILPSKPTTKQRQNKP QNKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTTKPTKK PTLKTTKKDPKPQTTKPKEVLTTKPTGKPTINTTKTNIRTTLLTSNTKGN PEHTSQEETLHSTTSEGYLSPSQVYTTSGQEETLHSTTSEGYLSPSQVYT TSEYLSQSLSSSNTTK

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 86, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 86.

An exemplary amino acid sequence of a recombinant RSV G comprising a RSV G ectodomain and transmembrane domain from RSV BA1, and a HPIV1 HN cytoplasmic tail is provided below:

(SEQ ID NO: 87) MAEKGKTNSSYWSTTRNDNSTVNTHINTPAGRTHWIAQIALSVLAMIIST SLIIAAIIFIISANHKVTLTTVTVQTIKNHTEKNITTYLTQVSPERVSPS KQLTTTPPIYTNSATISPNTKSETHHTTAQTKGRTTTPTQNNKPSTKPRP KNPPKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKP TNKPPTKTTNKRDPKKLAKTLKKETTINPTKKPTPKTTERDTSTSQSTVL DTTTSKHTERDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENTPNSTQTPT ASEPSTSNSTQKL

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 87, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 87.

An exemplary amino acid sequence of a recombinant RSV G comprising the RSV G ectodomain from RSV BA1, and HPIV1 HN transmembrane domain and cytoplasmic tail is provided below:

(SEQ ID NO: 88) MAEKGKENSSYWSTERNDNSTVNTHINTPAGRTHWILLIATTMHTVLSFI IMILCIDLIINHKVTLETVEVQTIKNHTEKNITTYLTQVSPERVSPSKQL ETTPPIYENSATISPNEKSETHHTTAQTKGRETTPTQNNKPSTKPRPKNP PKKPKDDYHFEVFNFVPCSICGNNQLCKSICKTIPSNKPKKKPTIKPENK PPEKTENKRDPKKLAKTLKKETTINPTKKPTPKTTERDTSTSQSTVLDTT ESKHTERDTSTSQSTVLDTTTSKHTIQQQSLHSTTPENTPNSTQTPTASE PSTSNSTQKL

In some embodiments, the heterologous gene encodes a recombinant RSV G comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 88, or an amino acid sequence at least 90% (such as at least 95% or at least 98%) identical to SEQ ID NO: 88.

In additional embodiments, the heterologous gene of the rB/HPIV3-RSV G comprises a sequence encoding a wild-type RSV G or variant thereof that has been codon-optimized for expression in a human cell. For example, the encoding sequence of the heterologous gene can be codon-optimized for human expression using a GeneArt (GA), DNA2.0 (D2), or GenScript (GS) optimization algorithm. Non-limiting examples of nucleic acid sequences encoding the RSV G protein that have been codon-optimized for expression in a human cell are provided as follows:

“GS” codon optimized DNA coding sequence for wt G from RSV A2: (SEQ ID NO: 89) atgtcaaagaacaaggatcagagaactgccaagaccctggaaagaacctg ggacaccctgaaccacctgctgtttatctcaagctgcctgtacaagctga atctgaaaagtgtggcccagatcaccctgtcaattctggctatgatcatt tcaacaagcctgatcattgccgctatcattttcatcgcaagcgccaacca caaggtcacccccaccacagctatcattcaggacgcaacatcccagatta agaacactacccccacctatctgacacagaatcctcagctgggaatctcc ccatctaacccctcagagattaccagccagatcacaactattctggcctc caccacacctggcgtgaagtccactctgcagtctactaccgtcaagacca aaaatacaactaccacacagacacagccttctaagccaactaccaaacag cggcagaataagccccctagtaaaccaaacaatgacttccattttgaggt gttcaactttgtcccatgcagcatctgttccaacaatcccacctgctggg ccatctgtaagagaattccaaacaagaaacccggcaagaagaccactacc aaacctactaagaaaccaaccctgaagacaactaagaaagatcctaaacc acagaccacaaagtctaaagaagtgcccactaccaagcctacagaggaac caactatcaacacaactaagactaacatcatcaccacactgctgacaagc aacactaccggcaatcccgagctgaccagccagatggaaacctttcactc cacaagctccgaggggaatcccagtccttcacaggtgtctacaactagtg aataccccagccagccttctagtccacccaacacccctaggcagtga Codon optimized (Biobasic) DNA coding sequence for wt G from RSV A/Maryland/001/11: (SEQ ID NO: 95) atgtctaagacaaaggatcagcggacagccaaaacactggaacggacatg ggataccctgaatcacctcctcttcatcagcagttgcctgtacaagctca atctgaagtccatcgcccagatcactctctccatccttgccatgatcatc tctacaagcctcatcattgccgcaattatcttcatcgccagcgctaacca caaggtcacccttaccacagccattattcaggatgccaccaaccagatca agaacacaacccctacctacctgacacagaaccctcagcttggaatttca ctgagcaacctgtccgaaaccacatctaaacctacaaccatcttggctct gaccacaccaaacgccgagtccaccccacaaagtaccacagtgaagacca aaaacaccacaaccacacagattcagccaagcaagcctacaactaagcaa aggcagaacaagccacagaacaaacccaacaacgactttcactttgaggt gttcaactttgtgccctgctccatttgctccaacaaccctacctgttggg ctatctgcaagaggatccccaacaagaagcccggcaggaagactactact aagcctactaaacagccagccattaagaccactaagaaggacccaaagcc acagacaaccaagccaaaggaggtgctcactaccaagcccactgagaagc ccaccattaacaccactaaaaccaacatccgcacaacattgctgacatca aacattacagagaaccaggagcacacaagccagaaggagacactgcatag cactacatccgaaggcaatcccagcccaagccaggtctatactacctcag agtacctgtcccagagcctgagccctagcaacactactagatggtag Codon optimized (Biobasic) DNA coding sequence for wt G from RSV Bl: (SEQ ID NO: 90) agtctaaacacaagaatcagcggaccgcccggaccttggaaaagacttgg gatacccttaaccaccttatcgtgatttcctcctgcctgtaccgcctcaa cctcaagagcattgctcagatcgcgctctcagtgctggccatgataatct ccacttccttgataattgccgccattatcttcattatttctgcaaaccac aaagtcaccctgaccaccgttaccgtgcaaaccattaaaaaccacacgga gaagaacatcactacatacctgactcaggttcccccggagcgagtgagca gctccaagcagcccacaacaacaagccctatccatacaaattcagcaaca acaagtccaaacacaaagtctgaaacgcatcacacaaccgctcagacgaa aggcaggaccacaacatccacccagactaataaacccagtactaagccta gactgaagaaccctcccaagaaacctaaggacgactatcatttcgaggtg tttaattttgtaccttgcagcatctgtggcaacaatcagctctgcaaaag catctgtaagaccatcccgtctaataagccaaagaagaagcccacgataa aaccaacaaataaaccaactaccaagacaacaaataagagggacccaaag acccccgctaaaactaccaagaaggagactaccaccaacccgacaaagaa acccaccctgacgactactgagagagatacttcaacttcacaaagcaccg tcctggatacaactaccctggagcacacaatccagcaacagagcctgcat agtactacgcctgaaaacactccaaactctacccagacgcccacagcctc agaaccttctacctccaatagtacccaaaatacccagagtcatgcatag

In some embodiments, the genome of the rB/HPIV3-RSV G vector comprises an antigenomic cDNA sequence set forth as any one of SEQ ID NOs: 85-88.

Non-limiting examples of methods of generating a recombinant parainfluenza virus (such as a 50 rB/HPIV3) including a heterologous gene, methods of attenuating the viruses (e.g., by recombinant or chemical means), as well as viral sequences and reagents for use in such methods are provided in US Patent Publications 2012/0045471, 2010/0119547, 2009/0263883, 2009/0017517, 7632508, 7622123, 7250171, 7208161, 7201907, 7192593, PCT Pub. No. WO 2016/118642, Liang et al. (J. Virol, 88(8): 4237-4250, 2014), and Tang et al. (J Virol, 77(20):10819-10828, 2003), each of which is incorporated by reference herein. In some embodiments, these methods can be modified as needed using the description provided herein to construct a disclosed rB/HPIV3-RSV G vector.

The genome of the rB/HPIV3-RSV G vector can include one or more variations (for example, mutations that cause an amino acid deletion, substitution, or insertion) as long as the resulting the rB/HPIV3-RSV G retains the desired biological function, such as a level of attenuation or immunogenicity. These variations in sequence can be naturally occurring variations or they can be engineered through the use of genetic engineering technique.

Other mutations involve replacement of the 3′ end of genome with its counterpart from antigenome, which is associated with changes in RNA replication and transcription. In addition, the intergenic regions (Collins et al., Proc. Natl. Acad. Sci. USA 83:4594-4598 (1986)) can be shortened or lengthened or changed in sequence content, and the naturally-occurring gene overlap (Collins et al., Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987)) can be removed or changed to a different intergenic region by the methods described herein.

In another embodiment, a sequence surrounding a translational start site (preferably including a nucleotide in the −3 position) of a selected viral gene is modified, alone or in combination with introduction of an upstream start codon, to modulate gene expression by specifying up- or down-regulation of translation.

Alternatively, or in combination with other modifications disclosed herein, gene expression can be modulated by altering a transcriptional GS signal of a selected gene(s) of the virus. In additional embodiments, modifications to a transcriptional GE signal can be incorporated into the viral genome.

In addition to the above described modifications to rB/HPIV3-RSV G, different or additional modifications to the genome can be made to facilitate manipulations, such as the insertion of unique restriction sites in various intergenic regions (e.g., a unique Asc I site between the N and P genes) or elsewhere. Nontranslated gene sequences can be removed to increase capacity for inserting foreign sequences.

Introduction of the foregoing modifications into rB/HPIV3-RSV G can be achieved by a variety of well-known methods. Examples of such techniques are found in see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual, 4^(th) ed., Cold Spring Harbor, N.Y., 2012) and Ausubel et al. (In Current Protocols in Molecular Biology, John Wiley & Sons, New York, through supplement 104, 2013). Thus, defined mutations can be introduced by conventional techniques (e.g., site-directed mutagenesis) into a cDNA copy of the genome or antigenome. The use of antigenome or genome cDNA subfragments to assemble a complete antigenome or genome cDNA has the advantage that each region can be manipulated separately (smaller cDNAs are easier to manipulate than large ones) and then readily assembled into a complete cDNA. Thus, the complete antigenome or genome cDNA, or any subfragment thereof, can be used as template for oligonucleotide-directed mutagenesis. A mutated subfragment can then be assembled into the complete antigenome or genome cDNA. Mutations can vary from single nucleotide changes to replacement of large cDNA pieces containing one or more genes or genome regions.

The disclosed embodiments of rB/HPIV3-RSV G are self-replicating, that is they are capable of replicating following infection of an appropriate host cell, and have an attenuated phenotype, for example when administered to a human subject. Preferably, the rB/HPIV3-RSV G is attenuated about 3- to 500-fold or more in the upper respiratory tract and about 100 to 5000 fold or more in the lower respiratory tract in a mammal compared to control HPIV3. In some embodiments, it is preferred that the level of viral replication in vitro is sufficient to provide for production of virus for use on a wide spread scale. In some embodiments, it is preferred that the level of viral replication of attenuated paramyxovirus in vitro is at least 10⁶, more preferably at least 10⁷, and most preferably at least 10⁸ per ml.

In some embodiments, the rB/HPIV3-RSV G vectors can be produced using the reverse genetics recombinant DNA-based technique (Collins, et al. 1995. Proc Natl Acad Sci USA 92:11563-11567). This system allows de novo recovery of infectious virus entirely from cDNA in a qualified cell substrate under defined conditions. Reverse genetics provides a means to introduce predetermined mutations into the rB/HPIV3-RSV G genome via the cDNA intermediate. Specific attenuating mutations were characterized in preclinical studies and combined to achieve the desired level of attenuation. Derivation of vaccine viruses from cDNA minimizes the risk of contamination with adventitious agents and helps to keep the passage history brief and well documented. Once recovered, the engineered virus strains propagate in the same manner as a biologically derived virus. As a result of passage and amplification, the virus does not contain recombinant DNA from the original recovery.

To propagate rB/HPIV3-RSV G vectors for immunization and other purposes, a number of cell lines which allow for viral growth may be used. Parainfluenza virus grows in a variety of human and animal cells. Preferred cell lines for propagating attenuated rB/HPIV3-RSV G virus for immunization include HEp-2 cells, FRhL-DBS2 cells, LLC-MK2 cells, MRC-5 cells, and Vero cells. Highest virus yields are usually achieved with epithelial cell lines such as Vero cells. Cells are typically inoculated with virus at a multiplicity of infection ranging from about 0.001 to 1.0, or more, and are cultivated under conditions permissive for replication of the virus, e.g., at about 30-37° C. and for about 3-10 days, or as long as necessary for virus to reach an adequate titer. Temperature-sensitive viruses often are grown using 32° C. as the “permissive temperature.” Virus is removed from cell culture and separated from cellular components, typically by standard clarification procedures, e.g., centrifugation, and may be further purified as desired using known procedures.

The rB/HPIV3-RSV G vectors can be tested in various well known and generally accepted in vitro and in vivo models to confirm adequate attenuation, resistance to phenotypic reversion, and immunogenicity. In in vitro assays, the modified virus is tested for temperature sensitivity of virus replication or “ts phenotype,” and for the small plaque phenotype. Modified virus also may be evaluated in an in vitro human airway epithelium (HAE) model, which appears to provide a means of ranking viruses in the order of their relative attenuation in non-human primates and humans (Zhang et al., 2002 J Virol 76:5654-5666; Schaap-Nutt et al., 2010 Vaccine 28:2788-2798; Ilyushina et al., 2012 J Virol 86:11725-11734). Modified viruses are further tested in animal models of HPIV3 or RSV infection. A variety of animal models (e.g., murine, cotton rat, and primate) are available.

Immunogenicity of a rB/HPIV3-RSV G vector can be assessed in an animal model (such as a non-human primate, for example an African green monkey), for example, by determining the number of animals that form antibodies to RSV and HPIV3 after one immunization and after a second immunization, and by measuring the magnitude of that response. In some embodiments, a rB/HPIV3-RSV G has sufficient immunogenicity if about 60 to 80% of the animals develop antibodies after the first immunization and about 80 to 100% of the animals develop antibodies after the second immunization. Preferably, the immune response protects against infection by both RSV and HPIV3.

Also provided are isolated polynucleotides comprising or consisting of the genome or antigenome of a disclosed rB/HPIV3-RSV G vector, vectors comprising the polynucleotides, and host cells comprising the polynucleotides or vectors.

IV. Immunogenic Compositions

Immunogenic compositions comprising a disclosed rB/HPIV3-RSV G vector and a pharmaceutically acceptable carrier are also provided. Such compositions can be administered to a subject by a variety of modes, for example, by an intranasal route. Standard methods for preparing administrable immunogenic compositions are described, for example, in such publications as Remingtons Pharmaceutical Sciences, 19^(th) Ed., Mack Publishing Company, Easton, Pa., 1995.

Potential carriers include, but are not limited to, physiologically balanced culture medium, phosphate buffer saline solution, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, cryoprotective additives or stabilizers such as proteins, peptides or hydrolysates (e.g., albumin, gelatin), sugars (e.g., sucrose, lactose, sorbitol), amino acids (e.g., sodium glutamate), or other protective agents. The resulting aqueous solutions may be packaged for use as is or lyophilized. Lyophilized preparations are combined with a sterile solution prior to administration for either single or multiple dosing.

The immunogenic composition can contain a bacteriostat to prevent or minimize degradation during storage, including but not limited to effective concentrations (usually ≤1% w/v) of benzyl alcohol, phenol, m-cresol, chlorobutanol, methylparaben, and/or propylparaben. A bacteriostat may be contraindicated for some patients; therefore, a lyophilized formulation may be reconstituted in a solution either containing or not containing such a component.

The immunogenic composition can contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate.

The immunogenic composition may optionally include an adjuvant to enhance the immune response of the host. Suitable adjuvants are, for example, toll-like receptor agonists, alum, AlPO4, alhydrogel, Lipid-A and derivatives or variants thereof, oil-emulsions, saponins, neutral liposomes, liposomes containing the recombinant virus, and cytokines, non-ionic block copolymers, and chemokines. Non-ionic block polymers containing polyoxyethylene (POE) and polyxylpropylene (POP), such as POE-POP-POE block copolymers, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.), among many other suitable adjuvants well known in the art, may be used as an adjuvant (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142). These adjuvants have the advantage in that they help to stimulate the immune system in a non-specific way, thus enhancing the immune response to a pharmaceutical product.

In some embodiments, the immunogenic composition can include a rB/HPIV3-RSV G encoding an RSV G ectodomain from one particular RSV subgroup or strain and also a recombinant rB/HPIV3-RSV G encoding an RSV G ectodomain from a different RSV subgroup or strain. For example, the composition can include rB/HPIV3-RSV G including recombinant RSV G proteins from subtype A and subtype B RSV. The different viruses can be in an admixture and administered simultaneously, or administered separately. Due to the phenomenon of cross-protection among certain strains of RSV, immunization with one rB/HPIV3-RSV G encoding a RSV G ectodomain from a first strain may protect against several different strains of the same or different subgroup.

In some instances it may be desirable to combine the immunogenic composition including the rB/HPIV3-RSV G, with other pharmaceutical products (e.g., vaccines) which induce protective responses to other viral agents, particularly those causing other childhood illnesses. For example, a composition including a rB/HPIV3-RSV G as described herein can also include other vaccines recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov/vaccines/acip/index.html) for the targeted age group (e.g., infants from approximately one to six months of age). These additional vaccines include, but are not limited to, IN-administered vaccines. As such, a rB/HPIV3-RSV G as described herein may be administered simultaneously with vaccines against, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcal bacteria (PCV), Haemophilus influenzae type b (Hib), polio, influenza and rotavirus.

In some embodiments, the immunogenic composition can be provided in unit dosage form for use to induce an immune response in a subject, for example, to prevent HPIV3 and/or RSV infection in the subject. A unit dosage form contains a suitable single preselected dosage for administration to a subject, or suitable marked or measured multiples of two or more preselected unit dosages, and/or a metering mechanism for administering the unit dose or multiples thereof.

V. Methods of Eliciting an Immune Response

Provided herein are methods of eliciting an immune response in a subject by administering an immunogenic composition containing a disclosed rB/HPIV3-RSV G to the subject. Upon immunization, the subject responds by producing antibodies specific for one or more of RSV G protein and HPIV3 HN and F proteins. In addition, innate and cell-mediated immune responses are induced, which can provide antiviral effectors as well as regulating the immune response. As a result of the immunization the host becomes at least partially or completely immune to HPIV3 and/or RSV infection, or resistant to developing moderate or severe HPIV3 and/or RSV disease, particularly of the lower respiratory tract.

Because nearly all humans are infected with RSV and HPIV3 by the age of 5, the entire birth cohort is included as a relevant population for immunization. This could be done, for example, by beginning an immunization regimen anytime from birth to 6 months of age, from 6 months of age to 5 years of age, in pregnant women (or women of child-bearing age) to protect their infants by passive transfer of antibody, family members of newborn infants or those still in utero, and subjects greater than 50 years of age. The scope of this disclosure is meant to include maternal immunization. In several embodiments, the subject is a human subject that is seronegative for RSV, HPIV3, and/or HPIV1 specific antibodies. In additional embodiments, the subject is no more than one year old, such as no more than 6 months old, no more than 3 months, or no more than 1 month old.

Subjects at greatest risk of RSV and/or HPIV infection with severe symptoms (e.g. requiring hospitalization) include children with prematurity, bronchopulmonary dysplasia, and congenital heart disease are most susceptible to severe disease. During childhood and adulthood, disease is milder but can be associated with lower airway disease and is commonly complicated by sinusitis. Disease severity increases in the institutionalized elderly (e.g., humans over 65 years old). Severe disease also occurs in persons with severe combined immunodeficiency disease or following bone marrow or lung transplantation. In some embodiments, these subjects can be selected for administration of a disclosed rB/HPIV3-RSV G.

The immunogenic compositions containing the rB/HPIV3-RSV G are administered to a subject susceptible to or otherwise at risk of RSV and/or HPIV3 infection in an “effective amount” which is sufficient to induce or enhance the individual's immune response capabilities against RSV and/or HPIV3. The immunogenic composition may be administered by any suitable method, including but not limited to, via injection, aerosol delivery, nasal spray, nasal droplets, oral inoculation, or topical application. In a preferred embodiment, the attenuated virus is administered according to established human intranasal administration protocols (e.g., as discussed in Karron et al. JID 191:1093-104, 2005). Briefly, adults or children are inoculated intranasally via droplet with an effective amount of the rB/HPIV3-RSV G, typically in a volume of 0.5 ml of a physiologically acceptable diluent or carrier. This has the advantage of simplicity and safety compared to parenteral immunization with a non-replicating virus. It also provides direct stimulation of local respiratory tract immunity, which plays a major role in resistance to RSV and HPIV3. Further, this mode of vaccination effectively bypasses the immunosuppressive effects of HPIV3- and RSV-specific maternally-derived serum antibodies, which typically are found in the very young. Also, while the parenteral administration of RSV antigens can sometimes be associated with immunopathologic complications, this has not been observed with a live virus.

In all subjects, the precise amount of immunogen administered and the timing and repetition of administration will be determined by various factors, including the patient's state of health and weight, the mode of administration, the nature of the formulation, etc. Dosages will generally range from about 3.0 log₁₀ to about 6.0 log₁₀ plaque forming units (“PFU”) or more of virus per patient, more commonly from about 4.0 log₁₀ to 5.0 log₁₀ PFU virus per patient. In one embodiment, about 5.0 log₁₀ to 6.0 log₁₀ PFU per patient may be administered during infancy, such as between 1 and 6 months of age, and one or more additional booster doses could be given 2-6 months or more later. In another embodiment, young infants could be given a dose of about 5.0 log₁₀ to 6.0 log₁₀ PFU per patient at approximately 2, 4, and 6 months of age, which is the recommended time of administration of a number of other childhood vaccines. In yet another embodiment, an additional booster dose could be administered at approximately 10-15 months of age.

The embodiments of rB/HPIV3-RSV G described herein, and immunogenic compositions thereof, are administered to a subject in an amount effective to induce or enhance an immune response against the HPIV3 and RSV antigens included in the rB/HPIV3-RSV G in the subject. An effective amount will allow some growth and proliferation of the virus, in order to produce the desired immune response, but will not produce viral-associated symptoms or illnesses. Based on the guidance provided herein and knowledge in the art, the proper amount of rB/HPIV3-RSV G to use for immunization cane determined.

A desired immune response is to inhibit subsequent infection with RSV and/or HPIV3. The RSV and/or HPIV3infection does not need to be completely inhibited for the method to be effective. For example, administration of an effective amount of a disclosed rB/HPIV3-RSV G can decrease subsequent RSV and/or HPIV3infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by RSV and/or HPIV3) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (prevention of detectable RSV and/or HPIV3infection), as compared to a suitable control.

Determination of effective dosages is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject, or that induce a desired response in the subject (such as a neutralizing immune response). Suitable models in this regard include, for example, murine, rat, hamster, cotton rat, bovine, ovine, porcine, feline, ferret, non-human primate, and other accepted animal model subjects known in the art. Alternatively, effective dosages can be determined using in vitro models (for example, immunologic and histopathologic assays). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the composition (for example, amounts that are effective to elicit a desired immune response or alleviate one or more symptoms of a targeted disease).

Administration of the rB/HPIV3-RSV G to a subject can elicit the production of an immune response that is protective against serious lower respiratory tract disease, such as pneumonia and bronchiolitis, or croup, when the subject is subsequently infected or re-infected with a wild-type RSV or HPIV3. While the naturally circulating virus is still capable of causing infection, particularly in the upper respiratory tract, there is a reduced possibility of rhinitis as a result of the immunization and a possible boosting of resistance by subsequent infection by wild-type virus. Following immunization, there are detectable levels of host engendered serum and secretory antibodies which are capable of neutralizing homologous (of the same subgroup) wild-type virus in vitro and in vivo. In many instances the host antibodies will also neutralize wild-type virus of a different, non-vaccine subgroup. To achieve higher levels of cross-protection, for example, against heterologous strains of another subgroup, subjects can be immunized with multiple immunogenic compositions that together comprise rB/HPIV3-RSV G with genomes encoding a RSV G proteins from at least one predominant strain of both RSV subgroups A and B.

An immunogenic composition including one or more of the disclosed rB/HPIV3-RSV G viruses can be used in coordinate (or prime-boost) immunization protocols or combinatorial formulations. It is contemplated that there can be several boosts, and that each boost can be a different disclosed immunogen. It is also contemplated in some examples that the boost may be the same immunogen as another boost, or the prime. In certain embodiments, novel combinatorial immunogenic compositions and coordinate immunization protocols employ separate immunogens or formulations, each directed toward eliciting an anti-viral immune response, such as an immune response to RSV and HPIV3 proteins. Separate immunogenic compositions that elicit the anti-viral immune response can be combined in a polyvalent immunogenic composition administered to a subject in a single immunization step, or they can be administered separately (in monovalent immunogenic compositions) in a coordinate (or prime-boost) immunization protocol.

The resulting immune response can be characterized by a variety of methods. These include taking samples of nasal washes or sera for analysis of RSV-specific antibodies, which can be detected by tests including, but not limited to, complement fixation, plaque neutralization, enzyme-linked immunosorbent assay, luciferase-immunoprecipitation assay, and flow cytometry. In addition, immune responses can be detected by assay of cytokines in nasal washes or sera, ELISPOT of immune cells from either source, quantitative RT-PCR or microarray analysis of nasal wash or serum samples, and restimulation of immune cells from nasal washes or serum by re-exposure to viral antigen in vitro and analysis for the production or display of cytokines, surface markers, or other immune correlates measures by flow cytometry or for cytotoxic activity against indicator target cells displaying RSV antigens. In this regard, individuals are also monitored for signs and symptoms of upper respiratory illness.

EXAMPLES

The following examples are provided to illustrate particular features of certain embodiments, but the scope of the claims should not be limited to those features exemplified.

Example 1 Identification of rB/HPIV3 Vectors Expressing RSV G or Variants Thereof for Induction of a Protective Immune Response to RSV and HPIV3

This example describes development, production, and evaluation of a rB/HPIV3 vector to express wt G protein of RSV A2 strain, and variants thereof, from the second gene position, between the vector N and P genes.

The vector backbone used in this example is a chimera of bovine PIV3 (Kansas strain) and human PIV3 (JS strain), called rB/HPIV3 (Schmidt et al., 2000, J. Virol. 74:8922-8929). This vector consists of BPIV3 in which the genes encoding the F and HN glycoproteins (the two PIV3 neutralization antigens and major protective antigens) have been replaced by their counterparts from HPIV3. HPIV3 and BPIV3 are very closely-related viruses, and the rB/HPIV3 chimera contains all of the PIV3 genes and is fully replication-competent, but is attenuated in rhesus monkeys and humans due to the BPIV3 backbone (Karron et al., 2012, Vaccine 30:3975-3981).

A version of the rB/HPIV3 with the wild type (wt) RSV F gene inserted in the second gene position (N-P), called MEDI-534, previously was evaluated in HPIV3- and RSV-seronegative infants and children (Bernstein et al., Ped. Infect. Dis., J. 31:109-114, 2012). MEDI-534 was attenuated and well-tolerated (as was the empty rB/HPIV3 vector in a different study in seronegative children, Karron et al., 2012, Vaccine 30:3975-3981). However, while all vaccine recipients seroconverted against HPIV3, only half developed detectable serum RSV-neutralizing antibodies analyzed by a micro-neutralization assay in the absence of added complement. There also was loss of RSV F protein expression by substantial proportions of vector that had been recovered in nasal washes from vaccinees (Yang et al., Vaccine 31:2822-2827, 2013). An advantage of the rB/HPIV3 vector system is that, because rB/HPIV3 expressing RSV wt F protein was shown to be safe and well-tolerated in seronegative children, as noted above (Bernstein et al., 2012, Pediatr. Infect. Dis. J. 31:109-114), versions with improved RSV inserts can be anticipated to be similarly well-tolerated, putting them on a fast track for clinical development.

The rB/HPIV3-RSV G vectors were designed so that the RSV G gene was flanked by BPIV3 transcription regulatory elements including a gene end (GE) signal copied from the N gene, a gene start (GS) signal copied from the P gene, and CTT trinucleotide intergenic regions (FIG. 1A). Additionally, the HN gene of the rB/HPIV3 includes 263T and 370P amino acid assignments, which results in virus that could be recovered and passaged with substantially reduced appearance of adventitious mutations to increase the efficiency of virus production, analysis, and manufacture.

RSV G and RSV G Variants

The RSV G protein is expressed during RSV infection in two forms. One is the full-length transmembrane form (mG), which is expressed on the cell surface and is packaged into the virus particle. The other form is an N-terminally-truncated, secreted form, sG. The full-length G protein (mG) is a type II protein that has an N-terminal cytoplasmic tail (CT, predicted to comprise amino acids 1-37 in strain A2, see FIG. 1B), a hydrophobic transmembrane domain (TM, comprising approximately amino acids 38-65, see FIG. 1B), and an ectodomain (comprising approximately amino acids 66-298). The sG form is produced by alternative translation initiation at the second AUG codon (M48) in the ORF, whose corresponding position in the protein lies within the TM domain (see FIG. 1B). The N-terminus is then subjected to intracellular proteolytic trimming that creates a new N-terminus at N66 (FIG. 1B).

The ectodomain of G consists of two large divergent domains that flank a short central conserved region at amino acids 164-186. The divergent domains have a high content of proline, alanine, threonine, and serine amino acids, and (for strain A2) an estimated four N-linked and 24-25 O-linked carbohydrate side chains. The central conserved domain contains a cysteine noose (i.e., a tight turn stabilized by two disulfide bonds) that bears a conserved CX3C motif (CWAIC, 182-186 aa of the A2 strain). The mG and sG forms are believed to be essentially the same with regard to glycosylation and protein structure except that mG forms a multimer that probably is a trimer or tetramer, whereas sG remains a monomer.

A CX3C domain also occurs in the chemokine called fractalkine (FIG. 1C), and the sequences flanking the CX3C domains in RSV G and fractalkine also share sequence relatedness (Tripp et al., Nature Immunol. 2:732-738[[m]], 2001). The G protein has been shown to mimic fractalkine in the ability to induce leukocyte chemotaxis in vitro (Tripp et al., 2001, Nature Immunol 2:732-738), and also binds to the fractalkine receptor CX3CR1, which can initiate RSV infection (Tripp et al., 2001, Nature Immunol 2:732-738; Johnson et al., 2015, PLoS Pathog. 11:e1005318). Like the G protein, fractalkine is expressed as a full-length transmembrane form and a truncated secreted form. For fractalkine, the full-length transmembrane form acts as an adhesion molecule that interacts with the fractalkine receptor CX3CR1 expressed on T cells, NK cells, and monocytes; and the secreted form acts as a chemoattractant for the same cell types. The role of the CX3C domain in RSV attachment seems straight-forward (i.e., allowing the virus to bind to cells expressing CX3CR1), but the effects of the CX3C domain and sG on host immunity remain unclear. In principle, ablation of the CX3C domain and/or the expression of sG would be expected to reduce the chemotactic influx of immune cells and might reduce disease, and this is supported by several studies with mutant RSVs (e.g., Maher et al., 2004, Microbes Infect. 6:1049-1055; Boyoglu-Barnum et al., 2017, J. Virol., 91(10): e02059-16), although there also are contradictory data (e.g. Harcourt et al., 2006, J. Immunol. 176:1600-1608). Other effects of G also have been described: e.g., sG was shown to act as an antigen decoy to reduce neutralization by antibodies, and also appeared to interfere with clearance of RSV by macrophages and complement (Bukreyev et al., J. Virol., 86(19): 10880-4, 2012). As another example, in vitro, the G protein interfered with human dendritic cell activation (Johnson et al., 2012, J. Virol. 86:1339-1347). In addition, the binding of RSV to CX3CR1 expressed on human neonatal regulatory B cells lead to a Th2-polarized response (Zhivaki et al., 2017, Immunity 46:301-314). Thus, the effects of the G protein, including its sG form and CX3C motif, on host immunity are incompletely understood and likely are complex.

RSV G and nine derivatives thereof are shown in FIG. 1A (constructs (i)-(x)), which are described below:

Constructs (i) is wt RSV G. The wt RSV G used in this example is from subgroup A2 and has the amino acid sequence set forth as:

(SEQ ID NO: 22) MSKNKDQRTAKTLERTWDTLNHLLFISSCLYKLNLKSVAQITLSILAMII STSLIIAAIIFIASANHKVTPTTAIIQDATSQIKNTTPTYLTQNPQLGIS PSNPSEITSQITTILASTTPGVKSTLQSTTVKTKNTTTTQTQPSKPTTKQ RQNKPPSKPNNDFHFEVFNFVPCSICSNNPTCWAICKRIPNKKPGKKTTT KPTKKPTLKTTKKDPKPQTTKSKEVPTTKPTEEPTINTTKTNIITTLLTS NTTGNPELTSQMETFHSTSSEGNPSPSQVSTTSEYPSQPSSPPNTPRQ

Constructs (ii) and (iii) in FIG. 1A encode the mG and sG forms of G protein. The mG construct was made by ablating expression of sG by a M48I mutation (FIG. 1B), and the sG construct was made by deletion of the first 47 codons of the G ORF so that codon M48 initiates the ORF (FIG. 1B); as described previously, the protein subsequently gets trimmed intracellularly to a major product with an N-terminus of N66 (Roberts et al., J. Virol., 68(7): 4538-4546, 1994, Teng et al., J. Virol. 289:283-296, 2001).

Constructs (iv) and (v) in FIG. 1A (G_B3CT and G_B3TMCT) are chimeric proteins that have the CT and TMCT of wt G replaced by those of the vector BPIV3 HN protein (see FIG. 1B) to promote efficient packaging of RSV G into the vector particles. It is believed that the presence in RSV G of CT or TMCT domains from the vector HN protein would promote homologous interactions with internal vector proteins such as M and N to facilitate the incorporation of RSV G into the viral particle. Identification of the sequence boundaries of the TM and CT of BPIV3 HN was determined by inspection and alignment with HPIV3 HN (FIG. 1B).

Constructs (vi) and (vii) in FIG. 1A (G_dCX3C and G_wCX4C) have mutations that ablate the CX3C motif in the G protein: specifically, G_dCX3C has a C186R mutation that changes the assignment of the second cysteine residue in the CX3C motif (FIG. 1C), and G_wCX4C has the insertion of an alanine residue between positions 185 and 186 that disrupts the spacing of the motif (FIG. 1C).

In constructs (viii) and (ix) in FIG. 1A, the C186R mutation also was made in combination with the B3CT and B3TMCT substitutions (G_dCX3C_B3CT and G_dCX3C_B3TMCT).

In addition, a construct encoding wt G was codon-optimized for human expression by GenScript (wt G/GS-opt, FIG. 1A, form x).

The range of RSV G variants allowed assessment of the effects of sG, the CX3C motif, the CT and TMCT mutations, and codon optimization on the immunogenicity of the RSV G protein. In addition, possible effects on the immune response to the rB/HPIV3 vector could also be assessed. In this context, PIV3 is a suitable surrogate for RSV because it is a related respiratory virus that has general similarities in epidemiology, tissue tropism, and disease. In addition, the RSV G insert is not needed for replication of the PIV vector, removing this confounding factor. Thus, effects on vector immunogenicity, as well as possible changes in immunologic restriction of vector replication, were assessed.

Full antigenomic cDNA sequences for rB/HPIV3-RSV wtG (construct (i)), rB/HPIV3-RSV wt G/GS-opt (construct (x)), rB/HPIV3-RSV G_B3TMCT (construct (v)), rB/HPIV3-RSV G_B3CT (construct (iv)) are provided in the exemplary sequences section below.

Virus Replication and RSV G Expression

The rB/HPIV3 vectors expressing various forms of RSV G replicated efficiently in LLC-MK2 cells (7.6-8.6 log₁₀ TCID₅₀/ml). No growth defect was observed with any construct.

The intracellular expression of RSV G by the various constructs was evaluated in Vero (FIGS. 2A and 2B) and LLC-MK2 (FIG. 3) cells by Western blot analysis. Cells were infected with each vector at a multiplicity of infection (MOI) of 10 TCID₅₀ (50% tissue culture infection doses) per cell, or by wt RSV at MOI of 3 plaque formation units (PFU) per cell. Cells were harvested at 24 h post-infection (p.i.) and cell lysates were prepared, subjected to gel electrophoresis under denaturing and reducing conditions, and analyzed by Western blotting using polyclonal antibodies against RSV and HPIV3.

Intracellular RSV G expressed by the rB/HPIV3 vector expressing wt RSV G (FIG. 2A, lane 2) and by wt RSV (lane 11) was detected as a predominant diffuse band of 90-120 kD (FIG. 2A), which corresponds to the fully glycosylated form, and as less abundant bands of 35-50 kD that represent processing intermediates of the G protein with incomplete O-glycosylation. Expression of the various G protein species described above by the mG construct was essentially the same as for wt G (FIGS. 2A and 2B, lane 4 versus 2). In contrast, the sG construct had only a trace amount of the large diffuse band, consistent with efficient secretion. The sG construct also had versions of the incompletely-glycosylated bands of the G protein that were reduced in size due to the N-terminal truncation. The CT and TMCT substitutions increased expression of the large G band by 50-80%, an effect that is unexplained (FIGS. 2A and 2B, lanes 5 and 6 versus 2). In contrast, CX3C ablation reduced the accumulation of the large G band by 20-50% percent, which appeared to be due at least in part to an increase in the accumulation of the 35-50 kD incompletely-glycosylated forms (FIGS. 2A and 2B, lanes 7 and 8 versus 2). The reduction in accumulation of the large G band associated with the CX3C mutations was compensated for when combined with the B3CT and B3TMCT substitutions (FIGS. 2A and 2B, lanes 9 and 10). There were no significant differences in the expression of the BPIV3 N protein between the constructs, including the empty vector (FIG. 2A), suggesting that none of the forms of the RSV G had much effect on vector gene expression in vitro. Intracellular expression of the RSV G and BPIV3 N protein in LLC-MK2 cells (FIGS. 3A and 3B) followed similar patterns as their expression in Vero cells.

Secretion of RSV G was evaluated from Vero cell cultures that were infected in parallel as described above and incubated for 48 h (FIG. 2C, 2D). The overlying medium was collected and clarified by centrifugation at 10,000×g for 60 min in order to remove debris as well as rB/HPIV3 virions. The clarified supernatants were then subjected to Western blot analysis with polyclonal antibodies against RSV and HPIV3. In the case of cells infected with rB/HPIV3 expressing wt G (FIG. 2C, lane 2) or infected with RSV (lane 11), the only form of G protein that was detected in the clarified, virus-depleted medium was the large, diffuse 90-120 kD species, indicating only the completely glycosylated form of G was secreted into medium (FIG. 2C, lanes 2 and 11). As expected, very little G protein was detected in the medium from cells infected with the mG construct (FIGS. 2C and 2D, lane 4), while sG protein produced by the sG construct was 70% more abundant than with the wt G construct (FIGS. 2C and 2D, lane 3 versus 2). Ablation of the CX3C by either mutation appeared to slightly decrease the expression of sG protein (FIGS. 2C and 2D, lanes 7 and 8), which likely was due to the overall reduced expression (FIGS. 2A and 2B, lanes 7 and 8). The B3CT and B3TMCT substitution completely abrogated the expression of sG protein (FIGS. 2C and 2D, lanes 5-6 and 9-10). This perhaps was not surprising in the case of B3TMCT, since the native M48 codon that normally is used to initiate synthesis of sG protein is located in the TM domain of G that was replaced with the TM domain from HPIV3 HN protein (although two other AUG codons are present in the HPIV3 HN TM domain that apparently were not utilized, FIG. 1B). In the case of the B3CT substitution, the native TM with its M48 codon is present, and it is not known why it was not utilized. One possibility is that the nucleotide sequence upstream of this region influences initiation at the M48 codon.

The stability of RSV G expression by the rB/HPIV3 constructs following passage in vitro was determined by a double-staining plaque assay (FIG. 4A), similar to assays described previously for rB/HPIV3 constructs expressing the RSV F protein (Liang et al., J. Virol., 89(18): 9499-9510, 2015). Briefly, Vero cells were infected with 10-fold serially diluted viral stocks and incubated under methyl cellulose overlay until plaques formed. Plaques were fixed and stained with primary rabbit anti-HPIV3 or goat anti-RSV antibodies followed by species-specific secondary antibodies conjugated to infrared dye (PIV3, green; RSV, red) (FIG. 4A). Co-expression of HPIV3 antigens and RSV G by a single PFU was visualized as yellow. All of the vector stocks expressing RSV G were found to have almost 100% of PFU expressing RSV G, with the exception that this could not be determined in the case of the sG construct because the rapid secretion of G into the medium precluded staining of the plaques for this protein (FIG. 4A, column 3).

Additionally, a double-plaque assay was performed using an RSV G MAb (131-2G) that is specific to the CX3C domain (FIG. 4B). This antibody bound to plaques for all of the versions of G in which the CX3C motif was intact (FIG. 4B lanes 2, 4, 5, 6) except for sG (lane 3), but did not bind to the versions with mutations in the CX3C motif (FIG. 4B, lanes 7-10). This indicated that ablation of CX3C motif disrupted the 131-2G binding epitope on RSV G, and was confirmation of the absence of an intact CX3C motif in these mutants.

RSV G Packaging

To evaluate the packaging efficiency of RSV G into rB/HPIV3 virions, empty vector and various vectors expressing versions of RSV G were propagated in LLC-MK2, and wt RSV was propagated in Vero cells. The medium overlying the cells was harvested, clarified by low speed centrifugation, and subjected to centrifugation on 30%-60% discontinuous sucrose gradients. For each construct, four micrograms of purified virus were analyzed by Western blot (FIG. 5A) and were quantified and normalized relative to wt G as 1.0. This showed that the unmodified wt G was detectable in vector virions as a very faint band (FIG. 5A, lane 2), and its packaging efficiency was less than 5% per μg of virion protein that of wt RSV (FIGS. 5A and 5B, lanes 2 and 6). Note that most of the G protein that was packaged in RSV virions grown in Vero cells had a lower molecular weight (50-60 kD) than the normally predominant 90-120 kD form. This was previously shown to arise due to cleavage by the cellular protease cathepsin L during long incubations in that particular cell line (Corry et al., 2015, J. Virol. 90:1311-1320). In the quantitation shown in FIG. 5B, both forms were quantified for wt RSV. The TMCT substitution from the BPIV3 HN protein enhanced the packaging efficiency of RSV G by 8.3-fold (FIGS. 5A and B, lanes 2 and 5), and the same magnitude of effect also was observed for the G_dCX3C_B3TMCT construct. The BPIV3 HN CT substitution alone only marginally increased the packaging of RSV G into vector virions (FIGS. 5A and 5B, lanes 2 and 4). As expected, sG protein was not packaged in the vector (FIG. 5A, lane 3).

The packaging of RSV G was also visualized by immune-gold labeling and transmission electron microscopy (TEM, FIG. 6). Purified virions prepared as described above (FIG. 5) were labeled by incubation with MAb 131-2G followed by anti-mouse IgG conjugated with gold particles. The specimens were imaged by TEM as previously described (Liang et al., J. Virol., 90(21): 10022-10038, 2016). The results showed that wt RSV virions had extensive antibody staining, indicative of a high density of packaging of the RSV G protein (FIGS. 6A and 6B). In contrast, virions of rB/HPIV3-wt G had only sporadic labeling (FIGS. 6C and 6D), and many virions had no detectable G. There was essentially no antibody binding to virions of vector expressing the sG protein (FIGS. 6E and 6F). The B3CT substitution appeared to slightly increase antibody binding (FIGS. 6G and 6H), while the B3TMCT substitution greatly improved the efficiency of antibody binding (FIGS. 6I and 6J). Empty vector virions were not labeled with the immune-gold particles (FIGS. 6K and 6L). Thus, the densities of RSV G displayed in TEM analysis were in agreement with the efficiency of G packaging quantified by Western blot.

In Vivo Replication, Immunogenicity and Protection

The replication, immunogenicity, and protective efficacy of the vectors were evaluated in a hamster model (FIGS. 7-9, respectively). To evaluate replication, hamsters in groups of six were immunized IN with 10⁵ TCID₅₀ of the indicated vector or 10⁶ PFU of wt RSV. Lungs and nasal turbinates were collected on day 5 after immunization, and the titers of the rB/HPIV3 vectors were determined by TCID₅₀ hemadsorption assays on LLC-MK2 cell monolayers, whereas RSV titers were determined by plaque assay on Vero monolayers. In the nasal turbinates, all of the vectors bearing an RSV G insert replicated to similar titers as the empty rB/HPIV3 vector (FIG. 7A), whereas in the lungs the vectors bearing an RSV G insert all were slightly more attenuated than the empty vector, although the differences were not significant (FIG. 7B). There were no significant differences between the replication of the various vectors expressing various forms of RSV G. Thus, for example, the expression of sG, or lack of expression of sG, or presence of the CX3C motif, or the absence of this motif, did not have an impact on the immunobiological milieu of the hamster lung sufficient to affect the replication of the rB/HPIV3 vector, which has the same tissue tropism as RSV (Zhang et al., J. Virol. 76:5654-5666, 2002; Zhang et al., J. Virol. 79:1113-1124, 2005) and shares many similarities in biology.

To evaluate immunogenicity, hamsters were immunized with vectors and wt RSV as described above, and sera were collected on days 0 and 28 post-immunization, In vitro 60% plaque reduction neutralization assays were carried out to quantify the serum RSV- and HPIV3-neutralizing antibody titers (FIG. 8).

Serum RSV-neutralizing antibody titers were determined in vitro on Vero cells by a 60%-plaque reduction neutralization titer (PRNT₆₀) assay using recombinant RSV that expresses the green fluorescent protein (RSV-GFP, Munir et al., J. Virol. 82:8780-8796, 2008). Assays were done in the presence or absence of added complement. The presence of complement can confer virus-lysis and steric-hindrance capabilities that give neutralization activity to otherwise non-neutralizing antibodies, and that can enhance the neutralization activity of poorly-neutralizing antibodies, whereas neutralization in the absence of complement depends on the direct capability of the antibodies (Yoder et al. 2004, J. Med. Virol. 72:688-694). Thus, assays done without complement provide a more stringent evaluation, and detect only those antibodies that can directly neutralize RSV (Liang et al., J. Virol., 89(18): 9499-9510, 2015).

When serum RSV-neutralizing antibody titers were evaluated by a neutralization assay performed with added complement, vectors expressing wt G, mG, G_B3CT and G_B3TMCT were shown to induce similarly high titers (>1:1024) that are not statistically different from that induced by a 10-fold higher (10⁶ PFU) of wt (i.e., not attenuated) RSV (FIG. 8A, lanes 2, 4-6 versus 11). In contrast, the sG construct did not induce detectable serum RSV neutralization antibodies (FIG. 8A, lane 3), despite its robust secretory expression (FIG. 2C, lane 3). Interestingly, CX3C ablation drastically reduced the RSV serum neutralization titers (FIG. 8A, lanes 7-10), indicating the role of the CX3C motif for the induction of RSV-neutralizing antibodies. Thus, ablating the expression of sG, or ablating the CX3C motif, did not increase the immunogenicity of the G protein, as would have been expected if these features interfered significantly with host immunity. On the contrary, the CX3C motif contributed substantially to inducing a strong RSV-neutralizing antibody response. To compare G-versus F-induced neutralization activity, sera of hamsters immunized with the same dose of rB/HPIV3 expressing unmodified wild-type (wt) RSV F from a previous study (Liang et al., J. Virol., 89(18): 9499-9510, 2015) were assayed in parallel (FIG. 8A, lane 12). The wt G induced slightly lower titers of RSV serum neutralization antibodies than wt F, but the difference was not statistically significant (FIG. 8A, lanes 2 and 12).

When serum HPIV3-neutralizing antibody titers (i.e., against the vector) were evaluated by an assay with added complement, all of the vectors expressing various forms of RSV G were similarly immunogenic (FIG. 8B, lanes 2-10), with the exception of the G_B3TMCT construct which induced a slightly but significantly lower level of serum HPIV3-neutralizing antibodies than the empty vector (FIG. 8B, lanes 1 and 6). The lower titer induced by the G_B3TMCT construct suggested that the enhanced packaging of RSV G by its TMCT substitution interfered with vector replication efficiency in vivo, perhaps by reducing the packaging of vector HN protein. Thus, the presence or absence of the sG protein or the CX3C motif in the expressed G protein did not have a significant effect on the antibody response against the vector.

Hamster sera were also assayed for neutralizing serum antibodies against a subgroup B strain of RSV (B1). While the neutralization activity against the B1 strain remained similarly high for wt RSV and wt F sera (FIG. 8C, lanes 11-12), the B1 cross-neutralization activities induced by RSV G constructs were substantially reduced (FIG. 8C, lanes 2 and 4-6), and disruption of the CX3C motif almost completely abolished the neutralization activity (FIG. 8C, lanes 7-10). This indicates that the G protein of RSV G A2 induced moderate titers of cross-subgroup neutralizing antibodies; furthermore, this was highly dependent upon the presence of CX3C motif.

To further characterize the role of the CX3C motif in the induction of RSV-neutralizing antibodies, an A2 RSV strain bearing a mutated CX3C (CWAIS) of G was used to analyze the serum RSV-neutralizing titers in the presence of added complement (FIG. 8D). While the serum neutralizing activity against the CX3C-mutated RSV induced by the four CX3C-ablation mutants (FIG. 8D, lanes 7-10) remained relatively low compared to wt RSV and vector expressing wt F (FIG. 8D, lanes 11 and 12), the neutralization activity in sera of wt G, mG, G_B3CT and G_B3TMCT against this CWAIS mutant were equally low (FIG. 8D, lanes 2 and 4-6). This was further confirmation that the CX3C domain is a major neutralization epitope of the G protein.

The titers of serum neutralizing antibodies, measured in the presence of complement, against wt RSV A2 (from FIG. 8A), wt RSV B1 (from FIG. 8C), and RSV A2 CWAIS (from FIG. 8D) are shown in Table 1. This further illustrates that ablation of sG (resulting in mG) did not result in increased immunogenicity; that packaging of G into the vector particle did not increase immunogenicity; and that ablation of the CX3C motif strongly reduced immunogenicity.

TABLE 1 The relative mean RSV serum neutralization titers induced by 10⁵ TCID₅₀ vectors compared to that induced by 10⁶ PFU wt RSV A2 in hamsters. All titers were determined in the presence of complement. Strains of RSV used in neutralization assays Viruses for RSV A2 immunization RSV A2 RSV B1 CWAIS Empty vector <4 <4 <4 sG <4 <4 <4 wtG 3040 226 52 mG 2077 152 16 G_B3CT 1746 125 25 G_B3TMCT 1911 70 25 G_dCX3C 40 <4 21 G_wCX4C 58 4 35 G_dCX3C_B3CT 83 6 42 G_dCX3C_B3TMCT 54 <4 36 wt RSV A2 6339 2521 4640 RSV F 6889 3040 6654

Serum RSV-neutralizing antibody titers were also evaluated by a neutralization assay performed in the absence of added complement (FIG. 8E) which provides a more stringent assessment of neutralization activity. The complement-independent assay showed that, while both wt RSV and vector-expressed wt F induced moderate titers of complement-independent serum RSV-neutralizing antibodies (FIG. 8E, lanes 11 and 12), the G constructs induced relatively low titers of serum antibodies capable of neutralizing RSV in the absence of complement in vitro (FIG. 8E, lanes 2-10). This result is offered with the caveat that Vero cells are deficient in the CX3CR1 receptor that may play a significant role in attachment in vivo (Johnson S M et al., 2015, PLoS Pathog., 11:e1005318), and therefore this Vero-based assay may fail to detect G-specific neutralizing antibodies. This may be resolved by a neutralization assay using an in vitro model of mucociliary human airway epithelium (HAE), which does express the CX3CR1 receptor (Johnson S M et al., 2015, PLoS Pathog., 11:e1005318), although HAE cultures are not very amenable to in vitro neutralization assays.

To measure protective efficacy, the hamsters that were immunized in the experiment in FIG. 8 were challenged IN with 10⁶ PFU of wt RSV on day 31 post-immunization. Hamsters were sacrificed 3 days later. Nasal turbinates and lungs were collected and homogenized for RSV plaque assay titration (FIG. 9). In the nasal turbinates, vector expressing wt G (FIG. 9A, lane 2) was significantly more protective than any of the other G constructs (lanes 3-10). In contrast, the sG construct was not protective (FIG. 9A, lane 3), correlating with its inability to induce RSV-neutralizing antibody (FIG. 9A, lane 3). Constructs mG, G_B3CT and G_B3TMCT induced substantial protection (FIG. 9A, lanes 4-6); while CX3C ablation (lanes 7 and 8) drastically reduced the protection compared to the unmodified wt G (lane 2), to a level that was not statistically different from sG and the empty vector (lanes 1 and 3). Enhanced packaging of the dCX3C mutant by B3CT and B3TMCT (FIG. 9A, lanes 9 and 10) appeared to slightly improve the protective efficacy to the level that was significantly better than sG and the empty vector (lanes 1 and 3). Only the wt RSV inoculation achieved complete protection in the nasal turbinates (FIG. 9A, lane 11). Superior protection with wt RSV was not unexpected, since it was administered at a 10-fold higher dose, it is not attenuated, it bears both the F and G neutralization antigens, and it expresses all of the viral antigens and thus would induce a broader cellular immune response. In studies with PIVs in hamsters, internal proteins in chimeric viruses have been shown to induce protection in a short term (1-2 month) challenge, but this wanes by four months (Tao T et al., 2000, Vaccine 18:1359-1366).

Vector-induced protection against RSV was much better in the lungs than in the nasal turbinates. The vectors expressing wt G, mG, G_B3CT, and B_B3TMCT conferred almost complete protection (FIG. 9B, lanes 2 and 4-6). But the vectors expressing any of the four CXC3 ablation mutants were less protective, with 1-3 hamsters per group not completely protected (FIG. 9B, lanes 7-10). As was observed in the nasal turbinates, the TMCT substitution exhibited some improvement of protection with the dCX3C construct (FIG. 9B, lane 10).

The titers of serum RSV-neutralizing antibodies on day 28 following the initial immunization (measured for individual animals in the presence of added complement, from FIG. 8A), were plotted versus the titers of challenge RSV in the nasal turbinates (from FIG. 9A) and lungs (from FIG. 9B) of the same individuals, resulting in the plots shown in FIGS. 9C and 9D, respectively. This showed that protection against challenge RSV replication in either the nasal turbinates or lungs, indicated by a reduction in challenge virus titer, was strongly correlated with the titer of serum complement-dependent RSV neutralizing antibodies (FIGS. 9C and D, respectively).

Codon-Optimized G ORF (wtG/GS-Opt)

A rB/HPIV3 construct expressing a RSV G ORF that was codon-optimized for human expression (GenScript), with no changes to amino acid coding (wtG/GS-opt, construct (x) in FIG. 1A), replicated in Vero and LLCMK-2 cells with a high level of efficiency similar to that of the other vectors. The intracellular expression of G protein by construct wtG/GS-opt in LLC-MK2 cells and Vero cells was evaluated by Western blot analysis. The LLC-MK2 and Vero cells were infected with rB/HPIV3 expressing wtG/GS-opt or wtG at a MOI of 10 TCID₅₀ per cell, or with wt RSV at an MOI of 3 PFU per cell. Cells were harvested at 24 hours post infection and cell lysates were prepared, subjected to gel electrophoresis under denaturing and reducing conditions, and analyzed by Western blotting using polyclonal antibodies against RSV (FIGS. 10A and B). This showed that the codon-optimized version was expressed approximately 2.2- to 2.3-fold more efficiently than the non-optimized wt ORF (FIGS. 10C and D.

The in vivo replication and immunogenicity of rB/HPIV3 expressing the codon-optimized G ORF was evaluated in parallel with rB/HPIV3 expressing the unmodified wt G ORF. Following the methods described in FIG. 7, hamsters in groups of nine were immunized IN with 10⁴ TCID₅₀ of the indicated vector or 10⁶ PFU of wt RSV. Sera were collected on day 28 after immunization. Hamsters were challenged intranasally on day 30 with 10⁶ PFU of wt RSV. Lungs and nasal turbinates were collected on day 3 after challenge. The titers of challenge RSV replication were determined by plaque assay on Vero monolayers. Serum RSV-neutralizing antibody titers were evaluated by plaque reduction neutralization in the presence of complement (FIG. 11). The serum RSV-neutralizing antibody titer induced by wild-type G (wt G) was slightly higher than that induced by wild-type F (wt F), although the difference was not significant (FIG. 11, lanes 2 and 3). But the codon-optimized wild-type G (wtG/GS-opt) induced a 2-fold higher titer of serum RSV-neutralizing antibodies than the wt F, which was statistically significant (FIG. 11, lanes 2 and 4). This indicated that GS codon-optimization of RSV G enhanced the immunogenicity of the rB/HPIV3-RSV G vector. Similar to their trend in the induction of serum RSV-neutralizing antibodies, the wt G and wt G/GS-opt conferred increased protection against RSV challenge in the nasal turbinates compared to wt F (FIG. 12A). In the lungs, wt G and wtG/GS-opt conferred complete protection, as with the wt RSV (FIG. 12B, lanes 3-5), while wt F was only partially protective (FIG. 12B, lane 2). These results indicated that rB/HPIV3 expressing wt G was more immunogenic and protective than rB/HPIV3 expressing wt F in hamsters; and the GenScript codon-optimization of RSV G enhanced its immunogenicity and protective efficacy.

Blocking RSV Infection of Ciliated Airway Epithelial Cells by RSV G-Induced Serum Antibodies

Vero cells are known to be deficient in the CX3CR1 surface protein that has been identified as a major receptor for G-mediated attachment in vivo (Johnson S M et al., 2015, PLoS Pathog., 11:e1005318). Thus, an important component of virus neutralization by G-induced antibodies might be missed by neutralization assays in Vero cells. To better evaluate the neutralization activity of G-induced serum antibodies, we performed a serum neutralization assay using an in vitro model of differentiated mucociliary human airway epithelium (HAE) that has been shown to be a close facsimile of in vivo HAE, and which does express CX3CR1 (Johnson S M et al., 2015, PLoS Pathog., 11:e1005318).

HAE cells were differentiated in culture at an air-liquid interface. Full differentiation was verified by the formation of ciliated cells and tight junctions imaged by confocal microscopy. Sera of immunized hamsters from the experiment shown in FIG. 8 were tested for the ability to block RSV infection in the HAE model. Equal aliquots of RSV-GFP (A2 strain) were pre-incubated with hamster serum in the absence of added complement, and then used to infect HAE cultures. The inocula was removed after infection and cells were cultured at the air-liquid interface. At 48 hours post infection, the GFP foci of infected cells were visualized and quantified (FIG. 13). In the uninfected culture, there was a background of approximately 6 foci per culture insert (FIG. 13, lane 1), likely due to autofluorescent cells. In the RSV-GFP infected culture, an average of approximately 200 (ranging from 53 to 518) GFP fluorescent foci were detected (FIG. 13, lane 2). Pre-incubation of RSV-GFP with sera from hamsters infected with the empty vector had no effect in RSV infection in HAE cells (FIG. 13, lane 3). Remarkably, the sera from hamsters immunized with wt G or wt RSV almost completely prevented the infection (FIG. 13, lanes 4 and 6). Surprisingly, the sera from hamsters immunized by the vector expressing wt RSV F displayed little inhibitory activity in this model; but the sera from hamsters immunized by the vector expressing a stabilized prefusion F (from a previous study and used here as a positive control for high-quality neutralizing antibody response) completed blocked the RSV infection (FIG. 13, lane 8). Interestingly, the sera of hamsters infected with the G_dCX3C construct were only partially protective compared with sera with wt G (FIG. 13, lanes 4 and 5). These results indicated that the wt RSV G induced high-quality neutralizing antibodies that can effectively prevent RSV infection in HAE in the absence of added complement. In addition, the integrity of the CX3C motif was important for the induction of serum neutralization antibodies by RSV G. Unexpectedly, in this assay, the neutralizing activity of sera raised against wt G was greater than for wt F, likely reflecting an effect on attachment. These results indicate antibodies to RSV G are particularly effective in neutralization of RSV in an in vitro model of HAE that closely resembles authentic HAE, and therefore the G protein may be an effective antigen to include in an RSV vaccine.

CONCLUSION

A number of studies have provided data indicating that the CX3C motif in the RSV G protein and expression of the soluble form of RSV G (sG) has various deleterious effects on host immune responses to RSV infection. This has been reported to include reduced dendritic cell activation (Johnson et al., 2012, J. Virol. 86:1339-1347), augmented inflammatory responses (Johnson et al., 1998, J. Virol. 72:2871-2880), inhibition of innate immunity (Shingai et al., 2008, Int. Immunol. 20:1169-1180; Polack et a12005, Proc. Natl. Acad. Sci. USA 102:8996-9001), and inhibition of immune cell responses (Chirkova et al., 2013, J. Virol. 87:13466-13479), among other activities. Based on these studies, it was expected that ablation of the CX3C motif and/or sG should make a safer, more immunogenic vaccine (e.g., Boyoglu-Barnum et al., “Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective,” J. Virol., 91(10): e02059-16, 2017; Chirkova et al., 2013, J. Virol. 87:13466-13479).

Surprisingly, data provided in the present example indicate that the mG construct (construct (ii), FIG. 1A), from which expression of sG was ablated, was modestly less immunogenic and protective than wt G protein, when expressed from the rB/HPIV3 vector. The finding that ablation of expression of the sG protein did not increase immunogenicity and protection was contrary to the postulated role of sG in impairing immune responses. Furthermore, the enhanced expression of sG on its own did not affect vector replication or vector immunogenicity. This was evident in comparing wtG (that expresses sG) with mG (that does not), and evaluating the effects of expression of sG protein alone (construct (iii), FIG. 1A), none of which affected vector replication or immunogenicity. Thus, there was no evidence that sG affected the host immune response in a manner that significantly affected RSV G immunogenicity and protective efficacy, or vector replication and immunogenicity.

Additionally, ablation of the CX3C motif greatly reduced the immunogenicity of RSV G (whether assayed with or without complement) and protective efficacy against RSV. Thus, contrary to the studies discussed above, this region of RSV G provides a positive effect on RSV immunogenicity and protective efficacy. Ablating the CX3C domain also did not significantly affect vector replication or immunogenicity. Thus, this motif did not alter the pulmonary immune milieu sufficient to affect vector replication or immunogenicity.

This example shows that features of the RSV G protein such as sG protein and the CX3C motif do not impair the immunogenicity and protective efficacy of the G protein expressed from a rB/HPIV3 vector. rB/HPIV3-based immunogens expressing wt G ectodomain were identified as preferred for inducing an immune response to RSV G. Surprisingly, the wt G construct induced titers of serum RSV-neutralizing antibodies assayed in the presence of complement that were not significantly different than those induced by wt RSV, even though RSV was administered at a 10-fold higher dose, is not attenuated, and bears both the F and G neutralization antigens. Additionally, the HAE assay (FIG. 13) showed that antibodies induced by wt G completely blocked infection in a model that closely mimics in vivo HAE, the predominant site of RSV replication in vivo, whereas antibodies induced by wt F did not. The RSV G protein is often considered to be a secondary neutralization and protective antigen compared to F. The present data revise this view and indicate that the G protein can improve neutralization (and likely protection). This suggests that the inclusion of vector expressing RSV G to an RSV immunization protocol improved neutralization and protection, a contribution that is not appreciated by conventional neutralization assays that do not involve the CX3CR receptor for RSV.

Exemplary antigenomic cDNA sequences: rB/HPIV3- RSV wt G (SEQ ID NO: 91): accaaacaagagaagagactggtttgggaatattaattcaaataaaaattaacttaggattaaagaactttaccgaaagg taaggggaaagaaatcctaagagcttagccatgttgagtctattcgacacattcagtgcgcgtaggcaggagaacataac gaaatcagctggtggggctgttattcccgggcaaaaaaacactgtgtctatatttgctcttggaccatcaataacagatg acaatgataaaatgacattggctcttctctttttgtctcattctttagacaatgaaaagcagcatgcgcaaagagctgga tttttagtttctctgttatcaatggcttatgccaacccagaattatatttaacatcaaatggtagtaatgcagatgttaa atatgttatctacatgatagagaaagacccaggaagacagaaatatggtgggtttgtcgtcaagactagagagatggttt atgaaaagacaactgattggatgttcgggagtgatcttgagtatgatcaagacaatatgttgcaaaatggtagaagcact tctacaatcgaggatcttgttcatacttttggatatccatcgtgtcttggagcccttataatccaagtttggataatact tgttaaggctataaccagtatatcaggattgaggaaaggattctttactcggttagaagcatttcgacaagatggaacag ttaaatccagtctagtgttgagcggtgatgcagtagaacaaattggatcaattatgaggtcccaacagagcttggtaaca ctcatggttgaaacactgataacaatgaacacaggcaggaatgatctgacaacaatagaaaagaatatacagattgtagg aaactacatcagagatgcaggtcttgcttcatttttcaacacaatcagatatggcattgagactagaatggcagctctaa ctctgtctacccttagaccggatatcaacagactcaaggcactgatcgagttatatctatcaaaggggccacgtgctcct tttatatgcattttgagagatcccgtgcatggtgagtttgcaccaggcaactatcctgccctctggagttatgcgatggg tgtagcagttgtacaaaacaaggccatgcaacagtatgtaacaggaaggtcttatctggatattgaaatgttccaacttg gtcaagcagtggcacgtgatgccgagtcgcagatgagttcaatattagaggatgaactgggggtcacacaagaagccaag caaagcttgaagaaacacatgaagaacatcagcagttcagatacaacctttcataagcctacagggggatcagccataga aatggcgatagatgaagaagcagggcagcctgaatccagaggagatcaggatcaaggagatgagcctcggtcatccatag ttccttatgcatgggcagacgaaaccgggaatgacaatcaaactgaatcaactacagaaattgacagcatcaaaactgaa caaagaaacatcagagacaggctgaacaaaagactcaacgagaaaaggaaacagagtgacccgagatcaactgacatcac aaacaacacaaatcaaactgaaatagatgatttgttcagtgcattcggaagcaactagtcacaaagagatgaccaggcgc gccaagtaagaaaaacttaggattaatggacctgcaggatgtccaaaaacaaggaccaacgcaccgctaagacattagaa aggacctgggacactctcaatcatttattattcatatcatcgtgcttatataagttaaatcttaaatctgtagcacaaat cacattatccattctggcaatgataatctcaacttcacttataattgcagccatcatattcatagcctcggcaaaccaca aagtcacaccaacaactgcaatcatacaagatgcaacaagccagatcaagaacacaaccccaacatacctcacccagaat cctcagcttggaatcagtccctctaatccgtctgaaattacatcacaaatcaccaccatactagcttcaacaacaccagg agtcaagtcaaccctgcaatccacaacagtcaagaccaaaaacacaacaacaactcaaacacaacccagcaagcccacca caaaacaacgccaaaacaaaccaccaagcaaacccaataatgattttcactttgaagtgttcaactttgtaccctgcagc atatgcagcaacaatccaacctgctgggctatctgcaaaagaataccaaacaaaaaaccaggaaagaaaaccactaccaa gcccacaaaaaaaccaaccctcaagacaaccaaaaaagatcccaaacctcaaaccactaaatcaaaggaagtacccacca ccaagcccacagaagagccaaccatcaacaccaccaaaacaaacatcataactacactactcacctccaacaccacagga aatccagaactcacaagtcaaatggaaaccttccactcaacttcctccgaaggcaatccaagcccttctcaagtctctac aacatccgagtacccatcacaaccttcatctccacccaacacaccacgccagtgatagctagcggcgcgccagcaacaag taagaaaaacttaggattaatggaaattatccaatccagagacggaaggacaaatccagaatccaaccacaactcaatca accaaagattcatggaagacaatgttcaaaacaatcaaatcatggattcttgggaagagggatcaggagataaatcatct gacatctcatcggccctcgacatcattgaattcatactcagcaccgactcccaagagaacacggcagacagcaatgaaat caacacaggaaccacaagacttagcacgacaatctaccaacctgaatccaaaacaacagaaacaagcaaggaaaatagtg gaccagctaacaaaaatcgacagtttggggcatcacacgaacgtgccacagagacaaaagatagaaatgttaatcaggag actgtacagggaggatataggagaggaagcagcccagatagtagaactgagactatggtcactcgaagaatctccagaag cagcccagatcctaacaatggaacccaaatccaggaagatattgattacaatgaagttggagagatggataaggactcta ctaagagggaaatgcgacaatttaaagatgttccagtcaaggtatcaggaagtgatgccattcctccaacaaaacaagat ggagacggtgatgatggaagaggcctggaatctatcagtacatttgattcaggatataccagtatagtgactgccgcaac actagatgacgaagaagaactccttatgaagaacaacaggccaagaaagtatcaatcaacaccccagaacagtgacaagg gaattaaaaaaggggttggaaggccaaaagacacagacaaacaatcatcaatattggactacgaactcaacttcaaagga tcgaagaagagccagaaaatcctcaaagccagcacgaatacaggagaaccaacaagaccacagaatggatcccaggggaa gagaatcacatcctggaacatcctcaacagcgagagcggcaatcgaacagaatcaacaaaccaaacccatcagacatcaa cctcgggacagaaccacacaatgggaccaagcagaacaacctccgaaccaaggatcaagacacaaaagacggatggaaag gaaagagaggacacagaagagagcactcgatttacagaaagggcgattacattattacagaatcttggtgtaatccaatc tgcagcaaaattagacctataccaagacaagagagttgtgtgtgtggcgaatgtcctaaacaatgcagatactgcatcaa agatagacttcctagcaggtttgatgataggagtgtcaatggatcatgataccaaattaaatcagattcagaacgagata ttaagtttgaaaactgatcttaaaaagatggatgaatcacatagaagactaattgagaatcaaaaagaacaattatcact gatcacatcattaatctcaaatcttaaaattatgacagagagaggagggaagaaggaccaaccagaacctagcgggagga catccatgatcaagacaaaagcaaaagaagagaaaataaagaaagtcaggtttgaccctcttatggaaacacagggcatc gagaaaaacatccctgacctctatagatcaatagagaaaacaccagaaaacgacacacagatcaaatcagaaataaacag attgaatgatgaatccaatgccactagattagtacctagaagaataagcagtacaatgagatcattaataataatcatta acaacagcaatttatcatcaaaagcaaagcaatcatacatcaacgaactcaagctctgcaagagtgacgaggaagtgtct gagttgatggacatgttcaatgaggatgtcagctcccagtaaaccgccaaccaagggtcaacaccaagaaaaccaatagc acaaaacagccaatcagagaccaccccaatacaccaaaccaatcaacacataacaaagatcgcggccgcatagatgatta agaaaaacttaggatgaaaggactaatcaatcctccgaaacaatgagcatcaccaactccacaatctacacattcccaga atcctctttctccgagaatggcaacatagagccgttaccactcaaggtcaatgaacagagaaaggccatacctcatatta gggttgtcaagataggagatccgcccaaacatggatccagatatctggatgtctttttactgggcttctttgagatggaa aggtcaaaagacaggtatgggagcataagtgatctagatgatgatccaagttacaaggtttgtggctctggatcattgcc acttgggttggctagatacaccggaaatgatcaggaactcctacaggctgcaaccaagctcgatatagaagtaagaagaa ctgtaaaggctacggagatgatagtttacactgtacaaaacatcaaacctgaactatatccatggtccagtagattaaga aaagggatgttatttgacgctaataaggttgcacttgctcctcaatgtcttccactagatagagggataaaattcagggt gatatttgtgaactgcacagcaattggatcaataactctattcaaaatccctaagtccatggcattgttatcattgccta atacaatatcaataaatctacaagtacatatcaaaacaggagttcagacagattccaaaggagtagttcagattctagat gaaaaaggtgaaaaatcactaaatttcatggttcatctcgggttgatcaaaaggaagatgggcagaatgtactcagttga atattgtaagcagaagatcgagaagatgagattattattctcattgggattagttggagggatcagcttccacgtcaacg caactggctctatatcaaagacattagcaagtcaattagcattcaaaagagaaatctgctatcccctaatggatctgaat ccacacttaaattcagttatatgggcatcatcagttgaaattacaagggtagatgcagttctccagccttcattacctgg cgaattcagatactacccaaacatcatagcaaaaggggtcgggaaaatcagacagtaaaatcaacaaccctgatatccac cggtgtattaagccgaagcaaataaaggataatcaaaaacttaggacaaaagaggtcaataccaacaactattagcagtc acactcgcaagaataagagagaagggaccaaaaaagtcaaataggagaaatcaaaacaaaaggtacagaacaccagaaca acaaaatcaaaacatccaactcactcaaaacaaaaattccaaaagagaccggcaacacaacaagcactgaacacaatgcc aacttcaatactgctaattattacaaccatgatcatggcatctttctgccaaatagatatcacaaaactacagcacgtag gtgtattggtcaacagtcccaaagggatgaagatatcacaaaactttgaaacaagatatctaattttgagcctcatacca aaaatagaagactctaactcttgtggtgaccaacagatcaagcaatacaagaagttattggatagactgatcatcccttt atatgatggattaagattacagaaagatgtgatagtaaccaatcaagaatccaatgaaaacactgatcccagaacaaaac gattctttggaggggtaattggaaccattgctctgggagtagcaacctcagcacaaattacagcggcagttgctctggtt gaagccaagcaggcaagatcagacatcgaaaaactcaaagaagcaattagggacacaaacaaagcagtgcagtcagttca gagctccataggaaatttaatagtagcaattaaatcagtccaggattatgttaacaaagaaatcgtgccatcgattgcga ggctaggttgtgaagcagcaggacttcaattaggaattgcattaacacagcattactcagaattaacaaacatatttggt gataacataggatcgttacaagaaaaaggaataaaattacaaggtatagcatcattataccgcacaaatatcacagaaat attcacaacatcaacagttgataaatatgatatctatgatctgttatttacagaatcaataaaggtgagagttatagatg ttgacttgaatgattactcaatcaccctccaagtcagactccctttattaactaggctgctgaacactcagatctacaaa gtagattccatatcatataacatccaaaacagagaatggtatatccctcttcccagccatatcatgacgaaaggggcatt tctaggtggagcagacgtcaaagaatgtatagaagcattcagcagctatatatgcccttctgatccaggatttgtattaa accatgaaatagagagctgcttatcaggaaacatatcccaatgtccaagaacaacggtcacatcagacattgttccaaga tatgcatttgtcaatggaggagtggttgcaaactgtataacaaccacctgtacatgcaacggaattggtaatagaatcaa tcaaccacctgatcaaggagtaaaaattataacacataaagaatgtagtacaataggtatcaacggaatgctgttcaata caaataaagaaggaactcttgcattctatacaccaaatgatataacactaaacaattctgttgcacttgatccaattgac atatcaatcgagctcaacaaggccaaatcagatctagaagaatcaaaagaatggataagaaggtcaaatcaaaaactaga ttctattggaaattggcatcaatctagcactacaatcataattattttgataatgatcattatattgtttataattaata taacgataattacaattgcaattaagtattacagaattcaaaagagaaatcgagtggatcaaaatgacaagccatatgta ctaacaaacaaataacatatctacagatcattagatattaaaattataaaaaacttaggagtaaagttacgcaatccaac tctactcatataattgaggaaggacccaatagacaaatccaaattcgagatggaatactggaagcataccaatcacggaa aggatgctggtaatgagctggagacgtctatggctactcatggcaacaagctcactaataagataatatacatattatgg acaataatcctggtgttattatcaatagtcttcatcatagtgctaattaattccatcaaaagtgaaaaggcccacgaatc attgctgcaagacataaataatgagtttatggaaattacagaaaagatccaaatggcatcggataataccaatgatctaa tacagtcaggagtgaatacaaggcttcttacaattcagagtcatgtccagaattacataccaatatcattgacacaacag atgtcagatcttaggaaattcattagtgaaattacaattagaaatgataatcaagaagtgctgccacaaagaataacaca tgatgtaggtataaaacctttaaatccagatgatttttggagatgcacgtctggtcttccatctttaatgaaaactccaa aaataaggttaatgccagggccgggattattagctatgccaacgactgttgatggctgtgttagaactccgtctttagtt ataaatgatctgatttatgcttatacctcaaatctaattactcgaggttgtcaggatataggaaaatcatatcaagtctt acagatagggataataactgtaaactcagacttggtacctgacttaaatcctaggatctctcatacctttaacataaatg acaataggaagtcatgttctctagcactcctaaatacagatgtatatcaactgtgttcaactcccaaagttgatgaaaga tcagattatgcatcatcaggcatagaagatattgtacttgatattgtcaattatgatggttcaatctcaacaacaagatt taagaataataacataagctttgatcaaccatatgctgcactatacccatctgttggaccagggatatactacaaaggca aaataatatttctcgggtatggaggtcttgaacatccaataaatgagaatgtaatctgcaacacaactgggtgccccggg aaaacacagagagactgtaatcaagcatctcatagtccatggttttcagataggaggatggtcaactccatcattgttgt tgacaaaggcttaaactcaattccaaaattgaaagtatggacgatatctatgcgacaaaattactgggggtcagaaggaa ggttacttctactaggtaacaagatctatatatatacaagatctacaagttggcatagcaagttacaattaggaataatt gatattactgattacagtgatataaggataaaatggacatggcataatgtgctatcaagaccaggaaacaatgaatgtcc atggggacattcatgtccagatggatgtataacaggagtatatactgatgcatatccactcaatcccacagggagcattg tgtcatctgtcatattagactcacaaaaatcgagagtgaacccagtcataacttactcaacagcaaccgaaagagtaaac gagctggccatcctaaacagaacactctcagctggatatacaacaacaagctgcattacacactataacaaaggatattg ttttcatatagtagaaataaatcataaaagcttaaacacatttcaacccatgttgttcaaaacagagattccaaaaagct gcagttaatcataattaaccataatatgcatcaatctatctataatacaagtatatgataagtaatcagcaatcagacaa tagacgtacggaaataataaaaaacttaggagaaaagtgtgcaagaaaaatggacaccgagtcccacagcggcacaacat ctgacattctgtaccctgaatgtcacctcaattctcctatagttaaaggaaagatagcacaactgcatacaataatgagt ttgcctcagccctacgatatggatgatgattcaatactgattattactagacaaaaaattaaactcaataaattagataa aagacaacggtcaattaggaaattaagatcagtcttaatggaaagagtaagtgatctaggtaaatatacctttatcagat atccagagatgtctagtgaaatgttccaattatgtatacccggaattaataataaaataaatgaattgctaagtaaagca agtaaaacatataatcaaatgactgatggattaagagatctatgggttactatactatcgaagttagcatcgaaaaatga tggaagtaattatgatatcaatgaagatattagcaatatatcaaatgttcacatgacttatcaatcagacaaatggtata atccattcaagacatggtttactattaagtatgacatgagaagattacaaaaagccaaaaatgagattacattcaatagg cataaagattataatctattagaagaccaaaagaatatattgctgatacatccagaactcgtcttaatattagataaaca aaattacaatgggtatataatgactcctgaattggtactaatgtattgtgatgtagttgaagggaggtggaatataagtt catgtgcaaaattggatcctaagttacaatcaatgtattataagggtaacaatttatgggaaataatagatggactattc tcgaccttaggagaaagaacatttgacataatatcactattagaaccacttgcattatcgctcattcaaacttatgaccc ggttaaacagctcaggggggcttttttaaatcacgtgttatcagaaatggaattaatatttgcagctgagtgtacaacag aggaaatacctaatgtggattatatagataaaattttagatgtgttcaaagaatcaacaatagatgaaatagcagaaatt ttctctttcttccgaacttttggacaccctccattagaggcgagtatagcagcagagaaagttagaaagtatatgtatac tgagaaatgcttgaaatttgatactatcaataaatgtcatgctattttttgtacaataattataaatggatatagagaaa gacatggtggtcaatggcctccagttacattacctgtccatgcacatgaatttatcataaatgcatacggatcaaattct gccatatcatatgagaatgctgtagattattataagagcttcataggaataaaatttgacaagtttatagagcctcaatt ggatgaagacttaactatttatatgaaagataaagcattatccccaaagaaatcaaactgggacacagtctatccagctt caaacctgttataccgcactaatgtgtctcatgattcacgaagattggttgaagtatttatagcagatagtaaatttgat ccccaccaagtattagattacgtagaatcaggatattggctggatgatcctgaatttaatatctcatatagtttaaaaga gaaagaaataaaacaagaaggtagactttttgcaaaaatgacatacaagatgagggctacacaagtattatcagaaacat tattggcgaataatatagggaaattcttccaagagaatgggatggttaaaggagaaattgaattactcaagagactaaca acaatatctatgtctggagttccgcggtataatgaggtatacaataattcaaaaagtcacacagaagaacttcaagctta taatgcaattagcagttccaatttatcttctaatcagaagtcaaagaagtttgaatttaaatctacagatatatacaatg atggatacgaaaccgtaagctgcttcttaacgacagatcttaaaaaatattgtttaaattggaggtatgaatcaacagct ttattcggtgatacttgtaatcagatatttgggttaaaggaattatttaattggctgcaccctcgccttgaaaagagtac aatatatgttggagatccttattgcccgccatcagatattgaacatttaccacttgatgaccatcctgattcaggatttt atgttcataatcctaaaggaggaatagaagggttttgccaaaagttatggacactcatatctatcagtgcaatacattta gcagctgtcaaaatcggtgtaagagttactgcaatggttcaaggggataatcaagccatagctgttaccacaagagtacc taataattatgattataaagttaagaaagagattgtttataaagatgtggtaagattttttgattccttgagagaggtga tggatgatctgggtcatgagctcaaactaaatgaaactataataagtagtaaaatgtttatatatagcaaaaggatatac tatgacggaagaatccttcctcaggcattaaaagcattgtctagatgtgttttttggtctgaaacaatcatagatgagac aagatcagcatcctcaaatctggctacatcgtttgcaaaggccattgagaatggctactcacctgtattgggatatgtat gctcaatcttcaaaaatatccaacagttgtatatagcgcttggaatgaatataaacccaactataacccaaaatattaaa gatcaatatttcaggaatattcattggatgcaatatgcctccttaatccctgctagtgtcggaggatttaattatatggc catgtcaaggtgttttgtcagaaacattggagatcctacagtcgctgcgttagccgatattaaaagatttataaaagcaa atttgttagatcgaggtgtcctttacagaattatgaatcaagaaccaggcgagtcttcttttttagactgggcctcagat ccctattcatgtaacttaccacaatctcaaaatataaccaccatgataaagaatataactgcaagaaatgtactacagga ctcaccaaacccattactatctggattatttacaagtacaatgatagaagaggatgaggaattagctgagttcctaatgg acaggagaataatcctcccaagagttgcacatgacattttagataattctcttactggaattaggaatgctatagctggt atgttggatacaacaaaatcactaattcgagtagggataagcagaggaggattaacctataacttattaagaaagataag caactatgatcttgtacaatatgagacacttagtaaaactttaagactaatagtcagtgacaagattaagtatgaagata tgtgctcagtagacctagccatatcattaagacaaaaaatgtggatgcatttatcaggaggaagaatgataaatggactt gaaactccagatcctttagagttactgtctggagtaataataacaggatctgaacattgtaggatatgttattcaactga aggtgaaagcccatatacatggatgtatttaccaggcaatcttaatataggatcagctgagacaggaatagcatcattaa gggtcccttactttggatcagttacagatgagagatctgaagcacaattagggtatatcaaaaatctaagcaaaccagct aaggctgctataagaatagcaatgatatatacttgggcatttgggaatgacgaaatatcttggatggaagcatcacagat tgcacaaacacgtgcaaactttacattggatagcttaaagattttgacaccagtgacaacatcaacaaatctatcacaca ggttaaaagatactgctactcagatgaaattttctagtacatcacttattagagtaagcaggttcatcacaatatctaat gataatatgtctattaaagaagcaaatgaaactaaagatacaaatcttatttatcaacaggtaatgttaacaggattaag tgtatttgaatatctatttaggttagaggagagtacaggacataaccctatggtcatgcatctacatatagaggatggat gttgtataaaagagagttacaatgatgagcatatcaatccggagtctacattagagttaatcaaataccctgagagtaat gaatttatatatgataaggaccctttaaaggatatagatctatcaaaattaatggttataagagatcattcttatacaat tgacatgaattactgggatgacacagatattgtacatgcaatatcaatatgtactgcagttacaatagcagatacaatgt cgcagctagatcgggataatcttaaggagctggttgtgattgcaaatgatgatgatattaacagtctgataactgaattt ctgaccctagatatactagtgtttctcaaaacatttggagggttactcgtgaatcaatttgcatataccctttatggatt gaaaatagaaggaagggatcccatttgggattatataatgagaacattaaaagacacctcacattcagtacttaaagtat tatctaatgcactatctcatccaaaagtgtttaagagattttgggattgtggagttttgaatcctatttatggtcctaat actgctagtcaagatcaagttaagcttgctctctcgatttgcgagtactccttggatctatttatgagagaatggttgaa tggagcatcacttgagatctatatctgtgatagtgacatggaaatagcaaatgacagaagacaagcatttctctcaagac atcttgcctttgtgtgttgtttagcagagatagcatcttttggaccaaatttattaaatctaacatatctagagagactt gatgaattaaaacaatacttagatctgaacatcaaagaagatcctactcttaaatatgtgcaagtatcaggactgttaat taaatcattcccctcaactgttacgtatgtaaggaaaactgcgattaagtatctgaggattcgtggtattaatccgcctg aaacgattgaagattgggatcccatagaagatgagaatatcttagacaatattgttaaaactgtaaatgacaattgcagt gataatcaaaagagaaataaaagtagttatttctggggattagctctaaagaattatcaagtcgtgaaaataagatccat aacgagtgattctgaagttaatgaagcttcgaatgttactacacatggaatgacacttcctcagggaggaagttatctat cacatcagctgaggttatttggagtaaacagtacaagttgtcttaaagctcttgaattatcacaaatcttaatgagggaa gttaaaaaagataaagatagactctttttaggagaaggagcaggagctatgttagcatgttatgatgctacactcggtcc tgcaataaattattataattctggtttaaatattacagatgtaattggtcaacgggaattaaaaatcttcccatcagaag tatcattagtaggtaaaaaactaggaaatgtaacacagattcttaatcgggtgagggtgttatttaatgggaatcccaat tcaacatggataggaaatatggaatgtgagagtttaatatggagtgaattaaatgataagtcaattggtttagtacattg tgacatggagggagcgataggcaaatcagaagaaactgttctacatgaacattatagtattattaggattacatatttaa tcggggatgatgatgttgtcctagtatcaaaaattataccaactattactccgaattggtctaaaatactctatctatac aagttgtattggaaggatgtaagtgtagtgtcccttaaaacatccaatcctgcctcaacagagctttatttaatttcaaa agatgcttactgtactgtaatggaacccagtaatcttgttttatcaaaacttaaaaggatatcatcaatagaagaaaata atctattaaagtggataatcttatcaaaaaggaagaataacgagtggttacagcatgaaatcaaagaaggagaaagggat tatgggataatgaggccatatcatacagcactgcaaatttttggattccaaattaacttaaatcacttagctagagaatt tttatcaactcctgatttaaccaacattaataatataattcaaagttttacaagaacaattaaagatgttatgttcgaat gggtcaatatcactcatgacaataaaagacataaattaggaggaagatataatctattcccgcttaaaaataaggggaaa ttaagattattatcacgaagattagtactaagctggatatcattatccttatcaaccagattactgacgggccgttttcc agatgaaaaatttgaaaatagggcacagaccggatatgtatcattggctgatattgatttagaatccttaaagttattat caagaaatattgtcaaaaattacaaagaacacataggattaatatcatactggtttttgaccaaagaggtcaaaatacta atgaagcttataggaggagtcaaactactaggaattcctaaacagtacaaagagttagaggatcgatcatctcagggtta tgaatatgataatgaatttgatattgattaatacataaaaacataaaataaaacacctattcctcacccattcacttcca acaaaatgaaaagtaagaaaaacatgtaatatatatataccaaacagagtttttctcttgtttggt rB/HPIV3-RSV wt G/GS-opt (SEQ ID NO: 92): accaaacaagagaagagactggtttgggaatattaattcaaataaaaattaacttaggattaaagaactttaccgaaagg taaggggaaagaaatcctaagagcttagccatgttgagtctattcgacacattcagtgcgcgtaggcaggagaacataac gaaatcagctggtggggctgttattcccgggcaaaaaaacactgtgtctatatttgctcttggaccatcaataacagatg acaatgataaaatgacattggctcttctctttttgtctcattctttagacaatgaaaagcagcatgcgcaaagagctgga tttttagtttctctgttatcaatggcttatgccaacccagaattatatttaacatcaaatggtagtaatgcagatgttaa atatgttatctacatgatagagaaagacccaggaagacagaaatatggtgggtttgtcgtcaagactagagagatggttt atgaaaagacaactgattggatgttcgggagtgatcttgagtatgatcaagacaatatgttgcaaaatggtagaagcact tctacaatcgaggatcttgttcatacttttggatatccatcgtgtcttggagcccttataatccaagtttggataatact tgttaaggctataaccagtatatcaggattgaggaaaggattctttactcggttagaagcatttcgacaagatggaacag ttaaatccagtctagtgttgagcggtgatgcagtagaacaaattggatcaattatgaggtcccaacagagcttggtaaca ctcatggttgaaacactgataacaatgaacacaggcaggaatgatctgacaacaatagaaaagaatatacagattgtagg aaactacatcagagatgcaggtcttgcttcatttttcaacacaatcagatatggcattgagactagaatggcagctctaa ctctgtctacccttagaccggatatcaacagactcaaggcactgatcgagttatatctatcaaaggggccacgtgctcct tttatatgcattttgagagatcccgtgcatggtgagtttgcaccaggcaactatcctgccctctggagttatgcgatggg tgtagcagttgtacaaaacaaggccatgcaacagtatgtaacaggaaggtcttatctggatattgaaatgttccaacttg gtcaagcagtggcacgtgatgccgagtcgcagatgagttcaatattagaggatgaactgggggtcacacaagaagccaag caaagcttgaagaaacacatgaagaacatcagcagttcagatacaacctttcataagcctacagggggatcagccataga aatggcgatagatgaagaagcagggcagcctgaatccagaggagatcaggatcaaggagatgagcctcggtcatccatag ttccttatgcatgggcagacgaaaccgggaatgacaatcaaactgaatcaactacagaaattgacagcatcaaaactgaa caaagaaacatcagagacaggctgaacaaaagactcaacgagaaaaggaaacagagtgacccgagatcaactgacatcac aaacaacacaaatcaaactgaaatagatgatttgttcagtgcattcggaagcaactagtcacaaagagatgaccaggcgc gccaagtaagaaaaacttaggattaatggacctgcaggatgtcaaagaacaaggatcagagaactgccaagaccctggaa agaacctgggacaccctgaaccacctgctgtttatctcaagctgcctgtacaagctgaatctgaaaagtgtggcccagat caccctgtcaattctggctatgatcatttcaacaagcctgatcattgccgctatcattttcatcgcaagcgccaaccaca aggtcacccccaccacagctatcattcaggacgcaacatcccagattaagaacactacccccacctatctgacacagaat cctcagctgggaatctccccatctaacccctcagagattaccagccagatcacaactattctggcctccaccacacctgg cgtgaagtccactctgcagtctactaccgtcaagaccaaaaatacaactaccacacagacacagccttctaagccaacta ccaaacagcggcagaataagccccctagtaaaccaaacaatgacttccattttgaggtgttcaactttgtcccatgcagc atctgttccaacaatcccacctgctgggccatctgtaagagaattccaaacaagaaacccggcaagaagaccactaccaa acctactaagaaaccaaccctgaagacaactaagaaagatcctaaaccacagaccacaaagtctaaagaagtgcccacta ccaagcctacagaggaaccaactatcaacacaactaagactaacatcatcaccacactgctgacaagcaacactaccggc aatcccgagctgaccagccagatggaaacctttcactccacaagctccgaggggaatcccagtccttcacaggtgtctac aactagtgaataccccagccagccttctagtccacccaacacccctaggcagtgatagctagcggcgcgccagcaacaag taagaaaaacttaggattaatggaaattatccaatccagagacggaaggacaaatccagaatccaaccacaactcaatca accaaagattcatggaagacaatgttcaaaacaatcaaatcatggattcttgggaagagggatcaggagataaatcatct gacatctcatcggccctcgacatcattgaattcatactcagcaccgactcccaagagaacacggcagacagcaatgaaat caacacaggaaccacaagacttagcacgacaatctaccaacctgaatccaaaacaacagaaacaagcaaggaaaatagtg gaccagctaacaaaaatcgacagtttggggcatcacacgaacgtgccacagagacaaaagatagaaatgttaatcaggag actgtacagggaggatataggagaggaagcagcccagatagtagaactgagactatggtcactcgaagaatctccagaag cagcccagatcctaacaatggaacccaaatccaggaagatattgattacaatgaagttggagagatggataaggactcta ctaagagggaaatgcgacaatttaaagatgttccagtcaaggtatcaggaagtgatgccattcctccaacaaaacaagat ggagacggtgatgatggaagaggcctggaatctatcagtacatttgattcaggatataccagtatagtgactgccgcaac actagatgacgaagaagaactccttatgaagaacaacaggccaagaaagtatcaatcaacaccccagaacagtgacaagg gaattaaaaaaggggttggaaggccaaaagacacagacaaacaatcatcaatattggactacgaactcaacttcaaagga tcgaagaagagccagaaaatcctcaaagccagcacgaatacaggagaaccaacaagaccacagaatggatcccaggggaa gagaatcacatcctggaacatcctcaacagcgagagcggcaatcgaacagaatcaacaaaccaaacccatcagacatcaa cctcgggacagaaccacacaatgggaccaagcagaacaacctccgaaccaaggatcaagacacaaaagacggatggaaag gaaagagaggacacagaagagagcactcgatttacagaaagggcgattacattattacagaatcttggtgtaatccaatc tgcagcaaaattagacctataccaagacaagagagttgtgtgtgtggcgaatgtcctaaacaatgcagatactgcatcaa agatagacttcctagcaggtttgatgataggagtgtcaatggatcatgataccaaattaaatcagattcagaacgagata ttaagtttgaaaactgatcttaaaaagatggatgaatcacatagaagactaattgagaatcaaaaagaacaattatcact gatcacatcattaatctcaaatcttaaaattatgacagagagaggagggaagaaggaccaaccagaacctagcgggagga catccatgatcaagacaaaagcaaaagaagagaaaataaagaaagtcaggtttgaccctcttatggaaacacagggcatc gagaaaaacatccctgacctctatagatcaatagagaaaacaccagaaaacgacacacagatcaaatcagaaataaacag attgaatgatgaatccaatgccactagattagtacctagaagaataagcagtacaatgagatcattaataataatcatta acaacagcaatttatcatcaaaagcaaagcaatcatacatcaacgaactcaagctctgcaagagtgacgaggaagtgtct gagttgatggacatgttcaatgaggatgtcagctcccagtaaaccgccaaccaagggtcaacaccaagaaaaccaatagc acaaaacagccaatcagagaccaccccaatacaccaaaccaatcaacacataacaaagatcgcggccgcatagatgatta agaaaaacttaggatgaaaggactaatcaatcctccgaaacaatgagcatcaccaactccacaatctacacattcccaga atcctctttctccgagaatggcaacatagagccgttaccactcaaggtcaatgaacagagaaaggccatacctcatatta gggttgtcaagataggagatccgcccaaacatggatccagatatctggatgtctttttactgggcttctttgagatggaa aggtcaaaagacaggtatgggagcataagtgatctagatgatgatccaagttacaaggtttgtggctctggatcattgcc acttgggttggctagatacaccggaaatgatcaggaactcctacaggctgcaaccaagctcgatatagaagtaagaagaa ctgtaaaggctacggagatgatagtttacactgtacaaaacatcaaacctgaactatatccatggtccagtagattaaga aaagggatgttatttgacgctaataaggttgcacttgctcctcaatgtcttccactagatagagggataaaattcagggt gatatttgtgaactgcacagcaattggatcaataactctattcaaaatccctaagtccatggcattgttatcattgccta atacaatatcaataaatctacaagtacatatcaaaacaggagttcagacagattccaaaggagtagttcagattctagat gaaaaaggtgaaaaatcactaaatttcatggttcatctcgggttgatcaaaaggaagatgggcagaatgtactcagttga atattgtaagcagaagatcgagaagatgagattattattctcattgggattagttggagggatcagcttccacgtcaacg caactggctctatatcaaagacattagcaagtcaattagcattcaaaagagaaatctgctatcccctaatggatctgaat ccacacttaaattcagttatatgggcatcatcagttgaaattacaagggtagatgcagttctccagccttcattacctgg cgaattcagatactacccaaacatcatagcaaaaggggtcgggaaaatcagacagtaaaatcaacaaccctgatatccac cggtgtattaagccgaagcaaataaaggataatcaaaaacttaggacaaaagaggtcaataccaacaactattagcagtc acactcgcaagaataagagagaagggaccaaaaaagtcaaataggagaaatcaaaacaaaaggtacagaacaccagaaca acaaaatcaaaacatccaactcactcaaaacaaaaattccaaaagagaccggcaacacaacaagcactgaacacaatgcc aacttcaatactgctaattattacaaccatgatcatggcatctttctgccaaatagatatcacaaaactacagcacgtag gtgtattggtcaacagtcccaaagggatgaagatatcacaaaactttgaaacaagatatctaattttgagcctcatacca aaaatagaagactctaactcttgtggtgaccaacagatcaagcaatacaagaagttattggatagactgatcatcccttt atatgatggattaagattacagaaagatgtgatagtaaccaatcaagaatccaatgaaaacactgatcccagaacaaaac gattctttggaggggtaattggaaccattgctctgggagtagcaacctcagcacaaattacagcggcagttgctctggtt gaagccaagcaggcaagatcagacatcgaaaaactcaaagaagcaattagggacacaaacaaagcagtgcagtcagttca gagctccataggaaatttaatagtagcaattaaatcagtccaggattatgttaacaaagaaatcgtgccatcgattgcga ggctaggttgtgaagcagcaggacttcaattaggaattgcattaacacagcattactcagaattaacaaacatatttggt gataacataggatcgttacaagaaaaaggaataaaattacaaggtatagcatcattataccgcacaaatatcacagaaat attcacaacatcaacagttgataaatatgatatctatgatctgttatttacagaatcaataaaggtgagagttatagatg ttgacttgaatgattactcaatcaccctccaagtcagactccctttattaactaggctgctgaacactcagatctacaaa gtagattccatatcatataacatccaaaacagagaatggtatatccctcttcccagccatatcatgacgaaaggggcatt tctaggtggagcagacgtcaaagaatgtatagaagcattcagcagctatatatgcccttctgatccaggatttgtattaa accatgaaatagagagctgcttatcaggaaacatatcccaatgtccaagaacaacggtcacatcagacattgttccaaga tatgcatttgtcaatggaggagtggttgcaaactgtataacaaccacctgtacatgcaacggaattggtaatagaatcaa tcaaccacctgatcaaggagtaaaaattataacacataaagaatgtagtacaataggtatcaacggaatgctgttcaata caaataaagaaggaactcttgcattctatacaccaaatgatataacactaaacaattctgttgcacttgatccaattgac atatcaatcgagctcaacaaggccaaatcagatctagaagaatcaaaagaatggataagaaggtcaaatcaaaaactaga ttctattggaaattggcatcaatctagcactacaatcataattattttgataatgatcattatattgtttataattaata taacgataattacaattgcaattaagtattacagaattcaaaagagaaatcgagtggatcaaaatgacaagccatatgta ctaacaaacaaataacatatctacagatcattagatattaaaattataaaaaacttaggagtaaagttacgcaatccaac tctactcatataattgaggaaggacccaatagacaaatccaaattcgagatggaatactggaagcataccaatcacggaa aggatgctggtaatgagctggagacgtctatggctactcatggcaacaagctcactaataagataatatacatattatgg acaataatcctggtgttattatcaatagtcttcatcatagtgctaattaattccatcaaaagtgaaaaggcccacgaatc attgctgcaagacataaataatgagtttatggaaattacagaaaagatccaaatggcatcggataataccaatgatctaa tacagtcaggagtgaatacaaggcttcttacaattcagagtcatgtccagaattacataccaatatcattgacacaacag atgtcagatcttaggaaattcattagtgaaattacaattagaaatgataatcaagaagtgctgccacaaagaataacaca tgatgtaggtataaaacctttaaatccagatgatttttggagatgcacgtctggtcttccatctttaatgaaaactccaa aaataaggttaatgccagggccgggattattagctatgccaacgactgttgatggctgtgttagaactccgtctttagtt ataaatgatctgatttatgcttatacctcaaatctaattactcgaggttgtcaggatataggaaaatcatatcaagtctt acagatagggataataactgtaaactcagacttggtacctgacttaaatcctaggatctctcatacctttaacataaatg acaataggaagtcatgttctctagcactcctaaatacagatgtatatcaactgtgttcaactcccaaagttgatgaaaga tcagattatgcatcatcaggcatagaagatattgtacttgatattgtcaattatgatggttcaatctcaacaacaagatt taagaataataacataagctttgatcaaccatatgctgcactatacccatctgttggaccagggatatactacaaaggca aaataatatttctcgggtatggaggtcttgaacatccaataaatgagaatgtaatctgcaacacaactgggtgccccggg aaaacacagagagactgtaatcaagcatctcatagtccatggttttcagataggaggatggtcaactccatcattgttgt tgacaaaggcttaaactcaattccaaaattgaaagtatggacgatatctatgcgacaaaattactgggggtcagaaggaa ggttacttctactaggtaacaagatctatatatatacaagatctacaagttggcatagcaagttacaattaggaataatt gatattactgattacagtgatataaggataaaatggacatggcataatgtgctatcaagaccaggaaacaatgaatgtcc atggggacattcatgtccagatggatgtataacaggagtatatactgatgcatatccactcaatcccacagggagcattg tgtcatctgtcatattagactcacaaaaatcgagagtgaacccagtcataacttactcaacagcaaccgaaagagtaaac gagctggccatcctaaacagaacactctcagctggatatacaacaacaagctgcattacacactataacaaaggatattg ttttcatatagtagaaataaatcataaaagcttaaacacatttcaacccatgttgttcaaaacagagattccaaaaagct gcagttaatcataattaaccataatatgcatcaatctatctataatacaagtatatgataagtaatcagcaatcagacaa tagacgtacggaaataataaaaaacttaggagaaaagtgtgcaagaaaaatggacaccgagtcccacagcggcacaacat ctgacattctgtaccctgaatgtcacctcaattctcctatagttaaaggaaagatagcacaactgcatacaataatgagt ttgcctcagccctacgatatggatgatgattcaatactgattattactagacaaaaaattaaactcaataaattagataa aagacaacggtcaattaggaaattaagatcagtcttaatggaaagagtaagtgatctaggtaaatatacctttatcagat atccagagatgtctagtgaaatgttccaattatgtatacccggaattaataataaaataaatgaattgctaagtaaagca agtaaaacatataatcaaatgactgatggattaagagatctatgggttactatactatcgaagttagcatcgaaaaatga tggaagtaattatgatatcaatgaagatattagcaatatatcaaatgttcacatgacttatcaatcagacaaatggtata atccattcaagacatggtttactattaagtatgacatgagaagattacaaaaagccaaaaatgagattacattcaatagg cataaagattataatctattagaagaccaaaagaatatattgctgatacatccagaactcgtcttaatattagataaaca aaattacaatgggtatataatgactcctgaattggtactaatgtattgtgatgtagttgaagggaggtggaatataagtt catgtgcaaaattggatcctaagttacaatcaatgtattataagggtaacaatttatgggaaataatagatggactattc tcgaccttaggagaaagaacatttgacataatatcactattagaaccacttgcattatcgctcattcaaacttatgaccc ggttaaacagctcaggggggcttttttaaatcacgtgttatcagaaatggaattaatatttgcagctgagtgtacaacag aggaaatacctaatgtggattatatagataaaattttagatgtgttcaaagaatcaacaatagatgaaatagcagaaatt ttctctttcttccgaacttttggacaccctccattagaggcgagtatagcagcagagaaagttagaaagtatatgtatac tgagaaatgcttgaaatttgatactatcaataaatgtcatgctattttttgtacaataattataaatggatatagagaaa gacatggtggtcaatggcctccagttacattacctgtccatgcacatgaatttatcataaatgcatacggatcaaattct gccatatcatatgagaatgctgtagattattataagagcttcataggaataaaatttgacaagtttatagagcctcaatt ggatgaagacttaactatttatatgaaagataaagcattatccccaaagaaatcaaactgggacacagtctatccagctt caaacctgttataccgcactaatgtgtctcatgattcacgaagattggttgaagtatttatagcagatagtaaatttgat ccccaccaagtattagattacgtagaatcaggatattggctggatgatcctgaatttaatatctcatatagtttaaaaga gaaagaaataaaacaagaaggtagactttttgcaaaaatgacatacaagatgagggctacacaagtattatcagaaacat tattggcgaataatatagggaaattcttccaagagaatgggatggttaaaggagaaattgaattactcaagagactaaca acaatatctatgtctggagttccgcggtataatgaggtatacaataattcaaaaagtcacacagaagaacttcaagctta taatgcaattagcagttccaatttatcttctaatcagaagtcaaagaagtttgaatttaaatctacagatatatacaatg atggatacgaaaccgtaagctgcttcttaacgacagatcttaaaaaatattgtttaaattggaggtatgaatcaacagct ttattcggtgatacttgtaatcagatatttgggttaaaggaattatttaattggctgcaccctcgccttgaaaagagtac aatatatgttggagatccttattgcccgccatcagatattgaacatttaccacttgatgaccatcctgattcaggatttt atgttcataatcctaaaggaggaatagaagggttttgccaaaagttatggacactcatatctatcagtgcaatacattta gcagctgtcaaaatcggtgtaagagttactgcaatggttcaaggggataatcaagccatagctgttaccacaagagtacc taataattatgattataaagttaagaaagagattgtttataaagatgtggtaagattttttgattccttgagagaggtga tggatgatctgggtcatgagctcaaactaaatgaaactataataagtagtaaaatgtttatatatagcaaaaggatatac tatgacggaagaatccttcctcaggcattaaaagcattgtctagatgtgttttttggtctgaaacaatcatagatgagac aagatcagcatcctcaaatctggctacatcgtttgcaaaggccattgagaatggctactcacctgtattgggatatgtat gctcaatcttcaaaaatatccaacagttgtatatagcgcttggaatgaatataaacccaactataacccaaaatattaaa gatcaatatttcaggaatattcattggatgcaatatgcctccttaatccctgctagtgtcggaggatttaattatatggc catgtcaaggtgttttgtcagaaacattggagatcctacagtcgctgcgttagccgatattaaaagatttataaaagcaa atttgttagatcgaggtgtcctttacagaattatgaatcaagaaccaggcgagtcttcttttttagactgggcctcagat ccctattcatgtaacttaccacaatctcaaaatataaccaccatgataaagaatataactgcaagaaatgtactacagga ctcaccaaacccattactatctggattatttacaagtacaatgatagaagaggatgaggaattagctgagttcctaatgg acaggagaataatcctcccaagagttgcacatgacattttagataattctcttactggaattaggaatgctatagctggt atgttggatacaacaaaatcactaattcgagtagggataagcagaggaggattaacctataacttattaagaaagataag caactatgatcttgtacaatatgagacacttagtaaaactttaagactaatagtcagtgacaagattaagtatgaagata tgtgctcagtagacctagccatatcattaagacaaaaaatgtggatgcatttatcaggaggaagaatgataaatggactt gaaactccagatcctttagagttactgtctggagtaataataacaggatctgaacattgtaggatatgttattcaactga aggtgaaagcccatatacatggatgtatttaccaggcaatcttaatataggatcagctgagacaggaatagcatcattaa gggtcccttactttggatcagttacagatgagagatctgaagcacaattagggtatatcaaaaatctaagcaaaccagct aaggctgctataagaatagcaatgatatatacttgggcatttgggaatgacgaaatatcttggatggaagcatcacagat tgcacaaacacgtgcaaactttacattggatagcttaaagattttgacaccagtgacaacatcaacaaatctatcacaca ggttaaaagatactgctactcagatgaaattttctagtacatcacttattagagtaagcaggttcatcacaatatctaat gataatatgtctattaaagaagcaaatgaaactaaagatacaaatcttatttatcaacaggtaatgttaacaggattaag tgtatttgaatatctatttaggttagaggagagtacaggacataaccctatggtcatgcatctacatatagaggatggat gttgtataaaagagagttacaatgatgagcatatcaatccggagtctacattagagttaatcaaataccctgagagtaat gaatttatatatgataaggaccctttaaaggatatagatctatcaaaattaatggttataagagatcattcttatacaat tgacatgaattactgggatgacacagatattgtacatgcaatatcaatatgtactgcagttacaatagcagatacaatgt cgcagctagatcgggataatcttaaggagctggttgtgattgcaaatgatgatgatattaacagtctgataactgaattt ctgaccctagatatactagtgtttctcaaaacatttggagggttactcgtgaatcaatttgcatataccctttatggatt gaaaatagaaggaagggatcccatttgggattatataatgagaacattaaaagacacctcacattcagtacttaaagtat tatctaatgcactatctcatccaaaagtgtttaagagattttgggattgtggagttttgaatcctatttatggtcctaat actgctagtcaagatcaagttaagcttgctctctcgatttgcgagtactccttggatctatttatgagagaatggttgaa tggagcatcacttgagatctatatctgtgatagtgacatggaaatagcaaatgacagaagacaagcatttctctcaagac atcttgcctttgtgtgttgtttagcagagatagcatcttttggaccaaatttattaaatctaacatatctagagagactt gatgaattaaaacaatacttagatctgaacatcaaagaagatcctactcttaaatatgtgcaagtatcaggactgttaat taaatcattcccctcaactgttacgtatgtaaggaaaactgcgattaagtatctgaggattcgtggtattaatccgcctg aaacgattgaagattgggatcccatagaagatgagaatatcttagacaatattgttaaaactgtaaatgacaattgcagt gataatcaaaagagaaataaaagtagttatttctggggattagctctaaagaattatcaagtcgtgaaaataagatccat aacgagtgattctgaagttaatgaagcttcgaatgttactacacatggaatgacacttcctcagggaggaagttatctat cacatcagctgaggttatttggagtaaacagtacaagttgtcttaaagctcttgaattatcacaaatcttaatgagggaa gttaaaaaagataaagatagactctttttaggagaaggagcaggagctatgttagcatgttatgatgctacactcggtcc tgcaataaattattataattctggtttaaatattacagatgtaattggtcaacgggaattaaaaatcttcccatcagaag tatcattagtaggtaaaaaactaggaaatgtaacacagattcttaatcgggtgagggtgttatttaatgggaatcccaat tcaacatggataggaaatatggaatgtgagagtttaatatggagtgaattaaatgataagtcaattggtttagtacattg tgacatggagggagcgataggcaaatcagaagaaactgttctacatgaacattatagtattattaggattacatatttaa tcggggatgatgatgttgtcctagtatcaaaaattataccaactattactccgaattggtctaaaatactctatctatac aagttgtattggaaggatgtaagtgtagtgtcccttaaaacatccaatcctgcctcaacagagctttatttaatttcaaa agatgcttactgtactgtaatggaacccagtaatcttgttttatcaaaacttaaaaggatatcatcaatagaagaaaata atctattaaagtggataatcttatcaaaaaggaagaataacgagtggttacagcatgaaatcaaagaaggagaaagggat tatgggataatgaggccatatcatacagcactgcaaatttttggattccaaattaacttaaatcacttagctagagaatt tttatcaactcctgatttaaccaacattaataatataattcaaagttttacaagaacaattaaagatgttatgttcgaat gggtcaatatcactcatgacaataaaagacataaattaggaggaagatataatctattcccgcttaaaaataaggggaaa ttaagattattatcacgaagattagtactaagctggatatcattatccttatcaaccagattactgacgggccgttttcc agatgaaaaatttgaaaatagggcacagaccggatatgtatcattggctgatattgatttagaatccttaaagttattat caagaaatattgtcaaaaattacaaagaacacataggattaatatcatactggtttttgaccaaagaggtcaaaatacta atgaagcttataggaggagtcaaactactaggaattcctaaacagtacaaagagttagaggatcgatcatctcagggtta tgaatatgataatgaatttgatattgattaatacataaaaacataaaataaaacacctattcctcacccattcacttcca acaaaatgaaaagtaagaaaaacatgtaatatatatataccaaacagagtttttctcttgtttggt rB/HPIV3- RSV G_B3TMCT (SEQ ID NO: 93): accaaacaagagaagagactggtttgggaatattaattcaaataaaaattaacttaggattaaagaactttaccgaaagg taaggggaaagaaatcctaagagcttagccatgttgagtctattcgacacattcagtgcgcgtaggcaggagaacataac gaaatcagctggtggggctgttattcccgggcaaaaaaacactgtgtctatatttgctcttggaccatcaataacagatg acaatgataaaatgacattggctcttctctttttgtctcattctttagacaatgaaaagcagcatgcgcaaagagctgga tttttagtttctctgttatcaatggcttatgccaacccagaattatatttaacatcaaatggtagtaatgcagatgttaa atatgttatctacatgatagagaaagacccaggaagacagaaatatggtgggtttgtcgtcaagactagagagatggttt atgaaaagacaactgattggatgttcgggagtgatcttgagtatgatcaagacaatatgttgcaaaatggtagaagcact tctacaatcgaggatcttgttcatacttttggatatccatcgtgtcttggagcccttataatccaagtttggataatact tgttaaggctataaccagtatatcaggattgaggaaaggattctttactcggttagaagcatttcgacaagatggaacag ttaaatccagtctagtgttgagcggtgatgcagtagaacaaattggatcaattatgaggtcccaacagagcttggtaaca ctcatggttgaaacactgataacaatgaacacaggcaggaatgatctgacaacaatagaaaagaatatacagattgtagg aaactacatcagagatgcaggtcttgcttcatttttcaacacaatcagatatggcattgagactagaatggcagctctaa ctctgtctacccttagaccggatatcaacagactcaaggcactgatcgagttatatctatcaaaggggccacgtgctcct tttatatgcattttgagagatcccgtgcatggtgagtttgcaccaggcaactatcctgccctctggagttatgcgatggg tgtagcagttgtacaaaacaaggccatgcaacagtatgtaacaggaaggtcttatctggatattgaaatgttccaacttg gtcaagcagtggcacgtgatgccgagtcgcagatgagttcaatattagaggatgaactgggggtcacacaagaagccaag caaagcttgaagaaacacatgaagaacatcagcagttcagatacaacctttcataagcctacagggggatcagccataga aatggcgatagatgaagaagcagggcagcctgaatccagaggagatcaggatcaaggagatgagcctcggtcatccatag ttccttatgcatgggcagacgaaaccgggaatgacaatcaaactgaatcaactacagaaattgacagcatcaaaactgaa caaagaaacatcagagacaggctgaacaaaagactcaacgagaaaaggaaacagagtgacccgagatcaactgacatcac aaacaacacaaatcaaactgaaatagatgatttgttcagtgcattcggaagcaactagtcacaaagagatgaccaggcgc gccaagtaagaaaaacttaggattaatggacctgcaggatggaatattggaaacacacaaacagcataaataacaccaac aatgaaaccgaaacagccagaggcaaacatagtagcaaggttacaaatatcataatgtacaccttctggacaataacatt aacaatattatcagtcatttttataatgatattgacaaacttaattaaccacaaagtcacaccaacaactgcaatcatac aagatgcaacaagccagatcaagaacacaaccccaacatacctcacccagaatcctcagcttggaatcagtccctctaat ccgtctgaaattacatcacaaatcaccaccatactagcttcaacaacaccaggagtcaagtcaaccctgcaatccacaac agtcaagaccaaaaacacaacaacaactcaaacacaacccagcaagcccaccacaaaacaacgccaaaacaaaccaccaa gcaaacccaataatgattttcactttgaagtgttcaactttgtaccctgcagcatatgcagcaacaatccaacctgctgg gctatctgcaaaagaataccaaacaaaaaaccaggaaagaaaaccactaccaagcccacaaaaaaaccaaccctcaagac aaccaaaaaagatcccaaacctcaaaccactaaatcaaaggaagtacccaccaccaagcccacagaagagccaaccatca acaccaccaaaacaaacatcataactacactactcacctccaacaccacaggaaatccagaactcacaagtcaaatggaa accttccactcaacttcctccgaaggcaatccaagcccttctcaagtctctacaacatccgagtacccatcacaaccttc atctccacccaacacaccacgccagtagtgatagctagcggcgcgccagcaacaagtaagaaaaacttaggattaatgga aattatccaatccagagacggaaggacaaatccagaatccaaccacaactcaatcaaccaaagattcatggaagacaatg ttcaaaacaatcaaatcatggattcttgggaagagggatcaggagataaatcatctgacatctcatcggccctcgacatc attgaattcatactcagcaccgactcccaagagaacacggcagacagcaatgaaatcaacacaggaaccacaagacttag cacgacaatctaccaacctgaatccaaaacaacagaaacaagcaaggaaaatagtggaccagctaacaaaaatcgacagt ttggggcatcacacgaacgtgccacagagacaaaagatagaaatgttaatcaggagactgtacagggaggatataggaga ggaagcagcccagatagtagaactgagactatggtcactcgaagaatctccagaagcagcccagatcctaacaatggaac ccaaatccaggaagatattgattacaatgaagttggagagatggataaggactctactaagagggaaatgcgacaattta aagatgttccagtcaaggtatcaggaagtgatgccattcctccaacaaaacaagatggagacggtgatgatggaagaggc ctggaatctatcagtacatttgattcaggatataccagtatagtgactgccgcaacactagatgacgaagaagaactcct tatgaagaacaacaggccaagaaagtatcaatcaacaccccagaacagtgacaagggaattaaaaaaggggttggaaggc caaaagacacagacaaacaatcatcaatattggactacgaactcaacttcaaaggatcgaagaagagccagaaaatcctc aaagccagcacgaatacaggagaaccaacaagaccacagaatggatcccaggggaagagaatcacatcctggaacatcct caacagcgagagcggcaatcgaacagaatcaacaaaccaaacccatcagacatcaacctcgggacagaaccacacaatgg gaccaagcagaacaacctccgaaccaaggatcaagacacaaaagacggatggaaaggaaagagaggacacagaagagagc actcgatttacagaaagggcgattacattattacagaatcttggtgtaatccaatctgcagcaaaattagacctatacca agacaagagagttgtgtgtgtggcgaatgtcctaaacaatgcagatactgcatcaaagatagacttcctagcaggtttga tgataggagtgtcaatggatcatgataccaaattaaatcagattcagaacgagatattaagtttgaaaactgatcttaaa aagatggatgaatcacatagaagactaattgagaatcaaaaagaacaattatcactgatcacatcattaatctcaaatct taaaattatgacagagagaggagggaagaaggaccaaccagaacctagcgggaggacatccatgatcaagacaaaagcaa aagaagagaaaataaagaaagtcaggtttgaccctcttatggaaacacagggcatcgagaaaaacatccctgacctctat agatcaatagagaaaacaccagaaaacgacacacagatcaaatcagaaataaacagattgaatgatgaatccaatgccac tagattagtacctagaagaataagcagtacaatgagatcattaataataatcattaacaacagcaatttatcatcaaaag caaagcaatcatacatcaacgaactcaagctctgcaagagtgacgaggaagtgtctgagttgatggacatgttcaatgag gatgtcagctcccagtaaaccgccaaccaagggtcaacaccaagaaaaccaatagcacaaaacagccaatcagagaccac cccaatacaccaaaccaatcaacacataacaaagatcgcggccgcatagatgattaagaaaaacttaggatgaaaggact aatcaatcctccgaaacaatgagcatcaccaactccacaatctacacattcccagaatcctctttctccgagaatggcaa catagagccgttaccactcaaggtcaatgaacagagaaaggccatacctcatattagggttgtcaagataggagatccgc ccaaacatggatccagatatctggatgtctttttactgggcttctttgagatggaaaggtcaaaagacaggtatgggagc ataagtgatctagatgatgatccaagttacaaggtttgtggctctggatcattgccacttgggttggctagatacaccgg aaatgatcaggaactcctacaggctgcaaccaagctcgatatagaagtaagaagaactgtaaaggctacggagatgatag tttacactgtacaaaacatcaaacctgaactatatccatggtccagtagattaagaaaagggatgttatttgacgctaat aaggttgcacttgctcctcaatgtcttccactagatagagggataaaattcagggtgatatttgtgaactgcacagcaat tggatcaataactctattcaaaatccctaagtccatggcattgttatcattgcctaatacaatatcaataaatctacaag tacatatcaaaacaggagttcagacagattccaaaggagtagttcagattctagatgaaaaaggtgaaaaatcactaaat ttcatggttcatctcgggttgatcaaaaggaagatgggcagaatgtactcagttgaatattgtaagcagaagatcgagaa gatgagattattattctcattgggattagttggagggatcagcttccacgtcaacgcaactggctctatatcaaagacat tagcaagtcaattagcattcaaaagagaaatctgctatcccctaatggatctgaatccacacttaaattcagttatatgg gcatcatcagttgaaattacaagggtagatgcagttctccagccttcattacctggcgaattcagatactacccaaacat catagcaaaaggggtcgggaaaatcagacagtaaaatcaacaaccctgatatccaccggtgtattaagccgaagcaaata aaggataatcaaaaacttaggacaaaagaggtcaataccaacaactattagcagtcacactcgcaagaataagagagaag ggaccaaaaaagtcaaataggagaaatcaaaacaaaaggtacagaacaccagaacaacaaaatcaaaacatccaactcac tcaaaacaaaaattccaaaagagaccggcaacacaacaagcactgaacacaatgccaacttcaatactgctaattattac aaccatgatcatggcatctttctgccaaatagatatcacaaaactacagcacgtaggtgtattggtcaacagtcccaaag ggatgaagatatcacaaaactttgaaacaagatatctaattttgagcctcataccaaaaatagaagactctaactcttgt ggtgaccaacagatcaagcaatacaagaagttattggatagactgatcatccctttatatgatggattaagattacagaa agatgtgatagtaaccaatcaagaatccaatgaaaacactgatcccagaacaaaacgattctttggaggggtaattggaa ccattgctctgggagtagcaacctcagcacaaattacagcggcagttgctctggttgaagccaagcaggcaagatcagac atcgaaaaactcaaagaagcaattagggacacaaacaaagcagtgcagtcagttcagagctccataggaaatttaatagt agcaattaaatcagtccaggattatgttaacaaagaaatcgtgccatcgattgcgaggctaggttgtgaagcagcaggac ttcaattaggaattgcattaacacagcattactcagaattaacaaacatatttggtgataacataggatcgttacaagaa aaaggaataaaattacaaggtatagcatcattataccgcacaaatatcacagaaatattcacaacatcaacagttgataa atatgatatctatgatctgttatttacagaatcaataaaggtgagagttatagatgttgacttgaatgattactcaatca ccctccaagtcagactccctttattaactaggctgctgaacactcagatctacaaagtagattccatatcatataacatc caaaacagagaatggtatatccctcttcccagccatatcatgacgaaaggggcatttctaggtggagcagacgtcaaaga atgtatagaagcattcagcagctatatatgcccttctgatccaggatttgtattaaaccatgaaatagagagctgcttat caggaaacatatcccaatgtccaagaacaacggtcacatcagacattgttccaagatatgcatttgtcaatggaggagtg gttgcaaactgtataacaaccacctgtacatgcaacggaattggtaatagaatcaatcaaccacctgatcaaggagtaaa aattataacacataaagaatgtagtacaataggtatcaacggaatgctgttcaatacaaataaagaaggaactcttgcat tctatacaccaaatgatataacactaaacaattctgttgcacttgatccaattgacatatcaatcgagctcaacaaggcc aaatcagatctagaagaatcaaaagaatggataagaaggtcaaatcaaaaactagattctattggaaattggcatcaatc tagcactacaatcataattattttgataatgatcattatattgtttataattaatataacgataattacaattgcaatta agtattacagaattcaaaagagaaatcgagtggatcaaaatgacaagccatatgtactaacaaacaaataacatatctac agatcattagatattaaaattataaaaaacttaggagtaaagttacgcaatccaactctactcatataattgaggaagga cccaatagacaaatccaaattcgagatggaatactggaagcataccaatcacggaaaggatgctggtaatgagctggaga cgtctatggctactcatggcaacaagctcactaataagataatatacatattatggacaataatcctggtgttattatca atagtcttcatcatagtgctaattaattccatcaaaagtgaaaaggcccacgaatcattgctgcaagacataaataatga gtttatggaaattacagaaaagatccaaatggcatcggataataccaatgatctaatacagtcaggagtgaatacaaggc ttcttacaattcagagtcatgtccagaattacataccaatatcattgacacaacagatgtcagatcttaggaaattcatt agtgaaattacaattagaaatgataatcaagaagtgctgccacaaagaataacacatgatgtaggtataaaacctttaaa tccagatgatttttggagatgcacgtctggtcttccatctttaatgaaaactccaaaaataaggttaatgccagggccgg gattattagctatgccaacgactgttgatggctgtgttagaactccgtctttagttataaatgatctgatttatgcttat acctcaaatctaattactcgaggttgtcaggatataggaaaatcatatcaagtcttacagatagggataataactgtaaa ctcagacttggtacctgacttaaatcctaggatctctcatacctttaacataaatgacaataggaagtcatgttctctag cactcctaaatacagatgtatatcaactgtgttcaactcccaaagttgatgaaagatcagattatgcatcatcaggcata gaagatattgtacttgatattgtcaattatgatggttcaatctcaacaacaagatttaagaataataacataagctttga tcaaccatatgctgcactatacccatctgttggaccagggatatactacaaaggcaaaataatatttctcgggtatggag gtcttgaacatccaataaatgagaatgtaatctgcaacacaactgggtgccccgggaaaacacagagagactgtaatcaa gcatctcatagtccatggttttcagataggaggatggtcaactccatcattgttgttgacaaaggcttaaactcaattcc aaaattgaaagtatggacgatatctatgcgacaaaattactgggggtcagaaggaaggttacttctactaggtaacaaga tctatatatatacaagatctacaagttggcatagcaagttacaattaggaataattgatattactgattacagtgatata aggataaaatggacatggcataatgtgctatcaagaccaggaaacaatgaatgtccatggggacattcatgtccagatgg atgtataacaggagtatatactgatgcatatccactcaatcccacagggagcattgtgtcatctgtcatattagactcac aaaaatcgagagtgaacccagtcataacttactcaacagcaaccgaaagagtaaacgagctggccatcctaaacagaaca ctctcagctggatatacaacaacaagctgcattacacactataacaaaggatattgttttcatatagtagaaataaatca taaaagcttaaacacatttcaacccatgttgttcaaaacagagattccaaaaagctgcagttaatcataattaaccataa tatgcatcaatctatctataatacaagtatatgataagtaatcagcaatcagacaatagacgtacggaaataataaaaaa cttaggagaaaagtgtgcaagaaaaatggacaccgagtcccacagcggcacaacatctgacattctgtaccctgaatgtc acctcaattctcctatagttaaaggaaagatagcacaactgcatacaataatgagtttgcctcagccctacgatatggat gatgattcaatactgattattactagacaaaaaattaaactcaataaattagataaaagacaacggtcaattaggaaatt aagatcagtcttaatggaaagagtaagtgatctaggtaaatatacctttatcagatatccagagatgtctagtgaaatgt tccaattatgtatacccggaattaataataaaataaatgaattgctaagtaaagcaagtaaaacatataatcaaatgact gatggattaagagatctatgggttactatactatcgaagttagcatcgaaaaatgatggaagtaattatgatatcaatga agatattagcaatatatcaaatgttcacatgacttatcaatcagacaaatggtataatccattcaagacatggtttacta ttaagtatgacatgagaagattacaaaaagccaaaaatgagattacattcaataggcataaagattataatctattagaa gaccaaaagaatatattgctgatacatccagaactcgtcttaatattagataaacaaaattacaatgggtatataatgac tcctgaattggtactaatgtattgtgatgtagttgaagggaggtggaatataagttcatgtgcaaaattggatcctaagt tacaatcaatgtattataagggtaacaatttatgggaaataatagatggactattctcgaccttaggagaaagaacattt gacataatatcactattagaaccacttgcattatcgctcattcaaacttatgacccggttaaacagctcaggggggcttt tttaaatcacgtgttatcagaaatggaattaatatttgcagctgagtgtacaacagaggaaatacctaatgtggattata tagataaaattttagatgtgttcaaagaatcaacaatagatgaaatagcagaaattttctctttcttccgaacttttgga caccctccattagaggcgagtatagcagcagagaaagttagaaagtatatgtatactgagaaatgcttgaaatttgatac tatcaataaatgtcatgctattttttgtacaataattataaatggatatagagaaagacatggtggtcaatggcctccag ttacattacctgtccatgcacatgaatttatcataaatgcatacggatcaaattctgccatatcatatgagaatgctgta gattattataagagcttcataggaataaaatttgacaagtttatagagcctcaattggatgaagacttaactatttatat gaaagataaagcattatccccaaagaaatcaaactgggacacagtctatccagcttcaaacctgttataccgcactaatg tgtctcatgattcacgaagattggttgaagtatttatagcagatagtaaatttgatccccaccaagtattagattacgta gaatcaggatattggctggatgatcctgaatttaatatctcatatagtttaaaagagaaagaaataaaacaagaaggtag actttttgcaaaaatgacatacaagatgagggctacacaagtattatcagaaacattattggcgaataatatagggaaat tcttccaagagaatgggatggttaaaggagaaattgaattactcaagagactaacaacaatatctatgtctggagttccg cggtataatgaggtatacaataattcaaaaagtcacacagaagaacttcaagcttataatgcaattagcagttccaattt atcttctaatcagaagtcaaagaagtttgaatttaaatctacagatatatacaatgatggatacgaaaccgtaagctgct tcttaacgacagatcttaaaaaatattgtttaaattggaggtatgaatcaacagctttattcggtgatacttgtaatcag atatttgggttaaaggaattatttaattggctgcaccctcgccttgaaaagagtacaatatatgttggagatccttattg cccgccatcagatattgaacatttaccacttgatgaccatcctgattcaggattttatgttcataatcctaaaggaggaa tagaagggttttgccaaaagttatggacactcatatctatcagtgcaatacatttagcagctgtcaaaatcggtgtaaga gttactgcaatggttcaaggggataatcaagccatagctgttaccacaagagtacctaataattatgattataaagttaa gaaagagattgtttataaagatgtggtaagattttttgattccttgagagaggtgatggatgatctgggtcatgagctca aactaaatgaaactataataagtagtaaaatgtttatatatagcaaaaggatatactatgacggaagaatccttcctcag gcattaaaagcattgtctagatgtgttttttggtctgaaacaatcatagatgagacaagatcagcatcctcaaatctggc tacatcgtttgcaaaggccattgagaatggctactcacctgtattgggatatgtatgctcaatcttcaaaaatatccaac agttgtatatagcgcttggaatgaatataaacccaactataacccaaaatattaaagatcaatatttcaggaatattcat tggatgcaatatgcctccttaatccctgctagtgtcggaggatttaattatatggccatgtcaaggtgttttgtcagaaa cattggagatcctacagtcgctgcgttagccgatattaaaagatttataaaagcaaatttgttagatcgaggtgtccttt acagaattatgaatcaagaaccaggcgagtcttcttttttagactgggcctcagatccctattcatgtaacttaccacaa tctcaaaatataaccaccatgataaagaatataactgcaagaaatgtactacaggactcaccaaacccattactatctgg attatttacaagtacaatgatagaagaggatgaggaattagctgagttcctaatggacaggagaataatcctcccaagag ttgcacatgacattttagataattctcttactggaattaggaatgctatagctggtatgttggatacaacaaaatcacta attcgagtagggataagcagaggaggattaacctataacttattaagaaagataagcaactatgatcttgtacaatatga gacacttagtaaaactttaagactaatagtcagtgacaagattaagtatgaagatatgtgctcagtagacctagccatat cattaagacaaaaaatgtggatgcatttatcaggaggaagaatgataaatggacttgaaactccagatcctttagagtta ctgtctggagtaataataacaggatctgaacattgtaggatatgttattcaactgaaggtgaaagcccatatacatggat gtatttaccaggcaatcttaatataggatcagctgagacaggaatagcatcattaagggtcccttactttggatcagtta cagatgagagatctgaagcacaattagggtatatcaaaaatctaagcaaaccagctaaggctgctataagaatagcaatg atatatacttgggcatttgggaatgacgaaatatcttggatggaagcatcacagattgcacaaacacgtgcaaactttac attggatagcttaaagattttgacaccagtgacaacatcaacaaatctatcacacaggttaaaagatactgctactcaga tgaaattttctagtacatcacttattagagtaagcaggttcatcacaatatctaatgataatatgtctattaaagaagca aatgaaactaaagatacaaatcttatttatcaacaggtaatgttaacaggattaagtgtatttgaatatctatttaggtt agaggagagtacaggacataaccctatggtcatgcatctacatatagaggatggatgttgtataaaagagagttacaatg atgagcatatcaatccggagtctacattagagttaatcaaataccctgagagtaatgaatttatatatgataaggaccct ttaaaggatatagatctatcaaaattaatggttataagagatcattcttatacaattgacatgaattactgggatgacac agatattgtacatgcaatatcaatatgtactgcagttacaatagcagatacaatgtcgcagctagatcgggataatctta aggagctggttgtgattgcaaatgatgatgatattaacagtctgataactgaatttctgaccctagatatactagtgttt ctcaaaacatttggagggttactcgtgaatcaatttgcatataccctttatggattgaaaatagaaggaagggatcccat ttgggattatataatgagaacattaaaagacacctcacattcagtacttaaagtattatctaatgcactatctcatccaa aagtgtttaagagattttgggattgtggagttttgaatcctatttatggtcctaatactgctagtcaagatcaagttaag cttgctctctcgatttgcgagtactccttggatctatttatgagagaatggttgaatggagcatcacttgagatctatat ctgtgatagtgacatggaaatagcaaatgacagaagacaagcatttctctcaagacatcttgcctttgtgtgttgtttag cagagatagcatcttttggaccaaatttattaaatctaacatatctagagagacttgatgaattaaaacaatacttagat ctgaacatcaaagaagatcctactcttaaatatgtgcaagtatcaggactgttaattaaatcattcccctcaactgttac gtatgtaaggaaaactgcgattaagtatctgaggattcgtggtattaatccgcctgaaacgattgaagattgggatccca tagaagatgagaatatcttagacaatattgttaaaactgtaaatgacaattgcagtgataatcaaaagagaaataaaagt agttatttctggggattagctctaaagaattatcaagtcgtgaaaataagatccataacgagtgattctgaagttaatga agcttcgaatgttactacacatggaatgacacttcctcagggaggaagttatctatcacatcagctgaggttatttggag taaacagtacaagttgtcttaaagctcttgaattatcacaaatcttaatgagggaagttaaaaaagataaagatagactc tttttaggagaaggagcaggagctatgttagcatgttatgatgctacactcggtcctgcaataaattattataattctgg tttaaatattacagatgtaattggtcaacgggaattaaaaatcttcccatcagaagtatcattagtaggtaaaaaactag gaaatgtaacacagattcttaatcgggtgagggtgttatttaatgggaatcccaattcaacatggataggaaatatggaa tgtgagagtttaatatggagtgaattaaatgataagtcaattggtttagtacattgtgacatggagggagcgataggcaa atcagaagaaactgttctacatgaacattatagtattattaggattacatatttaatcggggatgatgatgttgtcctag tatcaaaaattataccaactattactccgaattggtctaaaatactctatctatacaagttgtattggaaggatgtaagt gtagtgtcccttaaaacatccaatcctgcctcaacagagctttatttaatttcaaaagatgcttactgtactgtaatgga acccagtaatcttgttttatcaaaacttaaaaggatatcatcaatagaagaaaataatctattaaagtggataatcttat caaaaaggaagaataacgagtggttacagcatgaaatcaaagaaggagaaagggattatgggataatgaggccatatcat acagcactgcaaatttttggattccaaattaacttaaatcacttagctagagaatttttatcaactcctgatttaaccaa cattaataatataattcaaagttttacaagaacaattaaagatgttatgttcgaatgggtcaatatcactcatgacaata aaagacataaattaggaggaagatataatctattcccgcttaaaaataaggggaaattaagattattatcacgaagatta gtactaagctggatatcattatccttatcaaccagattactgacgggccgttttccagatgaaaaatttgaaaatagggc acagaccggatatgtatcattggctgatattgatttagaatccttaaagttattatcaagaaatattgtcaaaaattaca aagaacacataggattaatatcatactggtttttgaccaaagaggtcaaaatactaatgaagcttataggaggagtcaaa ctactaggaattcctaaacagtacaaagagttagaggatcgatcatctcagggttatgaatatgataatgaatttgatat tgattaatacataaaaacataaaataaaacacctattcctcacccattcacttccaacaaaatgaaaagtaagaaaaaca tgtaatatatatataccaaacagagtttttctcttgtttggt rB/HPIV3-RSV G_B3CT (SEQ ID NO: 94): accaaacaagagaagagactggtttgggaatattaattcaaataaaaattaacttaggattaaagaactttaccgaaagg taaggggaaagaaatcctaagagcttagccatgttgagtctattcgacacattcagtgcgcgtaggcaggagaacataac gaaatcagctggtggggctgttattcccgggcaaaaaaacactgtgtctatatttgctcttggaccatcaataacagatg acaatgataaaatgacattggctcttctctttttgtctcattctttagacaatgaaaagcagcatgcgcaaagagctgga tttttagtttctctgttatcaatggcttatgccaacccagaattatatttaacatcaaatggtagtaatgcagatgttaa atatgttatctacatgatagagaaagacccaggaagacagaaatatggtgggtttgtcgtcaagactagagagatggttt atgaaaagacaactgattggatgttcgggagtgatcttgagtatgatcaagacaatatgttgcaaaatggtagaagcact tctacaatcgaggatcttgttcatacttttggatatccatcgtgtcttggagcccttataatccaagtttggataatact tgttaaggctataaccagtatatcaggattgaggaaaggattctttactcggttagaagcatttcgacaagatggaacag ttaaatccagtctagtgttgagcggtgatgcagtagaacaaattggatcaattatgaggtcccaacagagcttggtaaca ctcatggttgaaacactgataacaatgaacacaggcaggaatgatctgacaacaatagaaaagaatatacagattgtagg aaactacatcagagatgcaggtcttgcttcatttttcaacacaatcagatatggcattgagactagaatggcagctctaa ctctgtctacccttagaccggatatcaacagactcaaggcactgatcgagttatatctatcaaaggggccacgtgctcct tttatatgcattttgagagatcccgtgcatggtgagtttgcaccaggcaactatcctgccctctggagttatgcgatggg tgtagcagttgtacaaaacaaggccatgcaacagtatgtaacaggaaggtcttatctggatattgaaatgttccaacttg gtcaagcagtggcacgtgatgccgagtcgcagatgagttcaatattagaggatgaactgggggtcacacaagaagccaag caaagcttgaagaaacacatgaagaacatcagcagttcagatacaacctttcataagcctacagggggatcagccataga aatggcgatagatgaagaagcagggcagcctgaatccagaggagatcaggatcaaggagatgagcctcggtcatccatag ttccttatgcatgggcagacgaaaccgggaatgacaatcaaactgaatcaactacagaaattgacagcatcaaaactgaa caaagaaacatcagagacaggctgaacaaaagactcaacgagaaaaggaaacagagtgacccgagatcaactgacatcac aaacaacacaaatcaaactgaaatagatgatttgttcagtgcattcggaagcaactagtcacaaagagatgaccaggcgc gccaagtaagaaaaacttaggattaatggacctgcaggatggaatattggaaacacacaaacagcataaataacaccaac aatgaaaccgaaacagccagaggcaaacatagtagcaaggttacaaatgtagcacaaatcacattatccattctggcaat gataatctcaacttcacttataattgcagccatcatattcatagcctcggcaaaccacaaagtcacaccaacaactgcaa tcatacaagatgcaacaagccagatcaagaacacaaccccaacatacctcacccagaatcctcagcttggaatcagtccc tctaatccgtctgaaattacatcacaaatcaccaccatactagcttcaacaacaccaggagtcaagtcaaccctgcaatc cacaacagtcaagaccaaaaacacaacaacaactcaaacacaacccagcaagcccaccacaaaacaacgccaaaacaaac caccaagcaaacccaataatgattttcactttgaagtgttcaactttgtaccctgcagcatatgcagcaacaatccaacc tgctgggctatctgcaaaagaataccaaacaaaaaaccaggaaagaaaaccactaccaagcccacaaaaaaaccaaccct caagacaaccaaaaaagatcccaaacctcaaaccactaaatcaaaggaagtacccaccaccaagcccacagaagagccaa ccatcaacaccaccaaaacaaacatcataactacactactcacctccaacaccacaggaaatccagaactcacaagtcaa atggaaaccttccactcaacttcctccgaaggcaatccaagcccttctcaagtctctacaacatccgagtacccatcaca accttcatctccacccaacacaccacgccagtagtgatagctagcggcgcgccagcaacaagtaagaaaaacttaggatt aatggaaattatccaatccagagacggaaggacaaatccagaatccaaccacaactcaatcaaccaaagattcatggaag acaatgttcaaaacaatcaaatcatggattcttgggaagagggatcaggagataaatcatctgacatctcatcggccctc gacatcattgaattcatactcagcaccgactcccaagagaacacggcagacagcaatgaaatcaacacaggaaccacaag acttagcacgacaatctaccaacctgaatccaaaacaacagaaacaagcaaggaaaatagtggaccagctaacaaaaatc gacagtttggggcatcacacgaacgtgccacagagacaaaagatagaaatgttaatcaggagactgtacagggaggatat aggagaggaagcagcccagatagtagaactgagactatggtcactcgaagaatctccagaagcagcccagatcctaacaa tggaacccaaatccaggaagatattgattacaatgaagttggagagatggataaggactctactaagagggaaatgcgac aatttaaagatgttccagtcaaggtatcaggaagtgatgccattcctccaacaaaacaagatggagacggtgatgatgga agaggcctggaatctatcagtacatttgattcaggatataccagtatagtgactgccgcaacactagatgacgaagaaga actccttatgaagaacaacaggccaagaaagtatcaatcaacaccccagaacagtgacaagggaattaaaaaaggggttg gaaggccaaaagacacagacaaacaatcatcaatattggactacgaactcaacttcaaaggatcgaagaagagccagaaa atcctcaaagccagcacgaatacaggagaaccaacaagaccacagaatggatcccaggggaagagaatcacatcctggaa catcctcaacagcgagagcggcaatcgaacagaatcaacaaaccaaacccatcagacatcaacctcgggacagaaccaca caatgggaccaagcagaacaacctccgaaccaaggatcaagacacaaaagacggatggaaaggaaagagaggacacagaa gagagcactcgatttacagaaagggcgattacattattacagaatcttggtgtaatccaatctgcagcaaaattagacct ataccaagacaagagagttgtgtgtgtggcgaatgtcctaaacaatgcagatactgcatcaaagatagacttcctagcag gtttgatgataggagtgtcaatggatcatgataccaaattaaatcagattcagaacgagatattaagtttgaaaactgat cttaaaaagatggatgaatcacatagaagactaattgagaatcaaaaagaacaattatcactgatcacatcattaatctc aaatcttaaaattatgacagagagaggagggaagaaggaccaaccagaacctagcgggaggacatccatgatcaagacaa aagcaaaagaagagaaaataaagaaagtcaggtttgaccctcttatggaaacacagggcatcgagaaaaacatccctgac ctctatagatcaatagagaaaacaccagaaaacgacacacagatcaaatcagaaataaacagattgaatgatgaatccaa tgccactagattagtacctagaagaataagcagtacaatgagatcattaataataatcattaacaacagcaatttatcat caaaagcaaagcaatcatacatcaacgaactcaagctctgcaagagtgacgaggaagtgtctgagttgatggacatgttc aatgaggatgtcagctcccagtaaaccgccaaccaagggtcaacaccaagaaaaccaatagcacaaaacagccaatcaga gaccaccccaatacaccaaaccaatcaacacataacaaagatcgcggccgcatagatgattaagaaaaacttaggatgaa aggactaatcaatcctccgaaacaatgagcatcaccaactccacaatctacacattcccagaatcctctttctccgagaa tggcaacatagagccgttaccactcaaggtcaatgaacagagaaaggccatacctcatattagggttgtcaagataggag atccgcccaaacatggatccagatatctggatgtctttttactgggcttctttgagatggaaaggtcaaaagacaggtat gggagcataagtgatctagatgatgatccaagttacaaggtttgtggctctggatcattgccacttgggttggctagata caccggaaatgatcaggaactcctacaggctgcaaccaagctcgatatagaagtaagaagaactgtaaaggctacggaga tgatagtttacactgtacaaaacatcaaacctgaactatatccatggtccagtagattaagaaaagggatgttatttgac gctaataaggttgcacttgctcctcaatgtcttccactagatagagggataaaattcagggtgatatttgtgaactgcac agcaattggatcaataactctattcaaaatccctaagtccatggcattgttatcattgcctaatacaatatcaataaatc tacaagtacatatcaaaacaggagttcagacagattccaaaggagtagttcagattctagatgaaaaaggtgaaaaatca ctaaatttcatggttcatctcgggttgatcaaaaggaagatgggcagaatgtactcagttgaatattgtaagcagaagat cgagaagatgagattattattctcattgggattagttggagggatcagcttccacgtcaacgcaactggctctatatcaa agacattagcaagtcaattagcattcaaaagagaaatctgctatcccctaatggatctgaatccacacttaaattcagtt atatgggcatcatcagttgaaattacaagggtagatgcagttctccagccttcattacctggcgaattcagatactaccc aaacatcatagcaaaaggggtcgggaaaatcagacagtaaaatcaacaaccctgatatccaccggtgtattaagccgaag caaataaaggataatcaaaaacttaggacaaaagaggtcaataccaacaactattagcagtcacactcgcaagaataaga gagaagggaccaaaaaagtcaaataggagaaatcaaaacaaaaggtacagaacaccagaacaacaaaatcaaaacatcca actcactcaaaacaaaaattccaaaagagaccggcaacacaacaagcactgaacacaatgccaacttcaatactgctaat tattacaaccatgatcatggcatctttctgccaaatagatatcacaaaactacagcacgtaggtgtattggtcaacagtc ccaaagggatgaagatatcacaaaactttgaaacaagatatctaattttgagcctcataccaaaaatagaagactctaac tcttgtggtgaccaacagatcaagcaatacaagaagttattggatagactgatcatccctttatatgatggattaagatt acagaaagatgtgatagtaaccaatcaagaatccaatgaaaacactgatcccagaacaaaacgattctttggaggggtaa ttggaaccattgctctgggagtagcaacctcagcacaaattacagcggcagttgctctggttgaagccaagcaggcaaga tcagacatcgaaaaactcaaagaagcaattagggacacaaacaaagcagtgcagtcagttcagagctccataggaaattt aatagtagcaattaaatcagtccaggattatgttaacaaagaaatcgtgccatcgattgcgaggctaggttgtgaagcag caggacttcaattaggaattgcattaacacagcattactcagaattaacaaacatatttggtgataacataggatcgtta caagaaaaaggaataaaattacaaggtatagcatcattataccgcacaaatatcacagaaatattcacaacatcaacagt tgataaatatgatatctatgatctgttatttacagaatcaataaaggtgagagttatagatgttgacttgaatgattact caatcaccctccaagtcagactccctttattaactaggctgctgaacactcagatctacaaagtagattccatatcatat aacatccaaaacagagaatggtatatccctcttcccagccatatcatgacgaaaggggcatttctaggtggagcagacgt caaagaatgtatagaagcattcagcagctatatatgcccttctgatccaggatttgtattaaaccatgaaatagagagct gcttatcaggaaacatatcccaatgtccaagaacaacggtcacatcagacattgttccaagatatgcatttgtcaatgga ggagtggttgcaaactgtataacaaccacctgtacatgcaacggaattggtaatagaatcaatcaaccacctgatcaagg agtaaaaattataacacataaagaatgtagtacaataggtatcaacggaatgctgttcaatacaaataaagaaggaactc ttgcattctatacaccaaatgatataacactaaacaattctgttgcacttgatccaattgacatatcaatcgagctcaac 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aatgactcctgaattggtactaatgtattgtgatgtagttgaagggaggtggaatataagttcatgtgcaaaattggatc ctaagttacaatcaatgtattataagggtaacaatttatgggaaataatagatggactattctcgaccttaggagaaaga acatttgacataatatcactattagaaccacttgcattatcgctcattcaaacttatgacccggttaaacagctcagggg ggcttttttaaatcacgtgttatcagaaatggaattaatatttgcagctgagtgtacaacagaggaaatacctaatgtgg attatatagataaaattttagatgtgttcaaagaatcaacaatagatgaaatagcagaaattttctctttcttccgaact tttggacaccctccattagaggcgagtatagcagcagagaaagttagaaagtatatgtatactgagaaatgcttgaaatt tgatactatcaataaatgtcatgctattttttgtacaataattataaatggatatagagaaagacatggtggtcaatggc ctccagttacattacctgtccatgcacatgaatttatcataaatgcatacggatcaaattctgccatatcatatgagaat gctgtagattattataagagcttcataggaataaaatttgacaagtttatagagcctcaattggatgaagacttaactat ttatatgaaagataaagcattatccccaaagaaatcaaactgggacacagtctatccagcttcaaacctgttataccgca ctaatgtgtctcatgattcacgaagattggttgaagtatttatagcagatagtaaatttgatccccaccaagtattagat tacgtagaatcaggatattggctggatgatcctgaatttaatatctcatatagtttaaaagagaaagaaataaaacaaga aggtagactttttgcaaaaatgacatacaagatgagggctacacaagtattatcagaaacattattggcgaataatatag ggaaattcttccaagagaatgggatggttaaaggagaaattgaattactcaagagactaacaacaatatctatgtctgga gttccgcggtataatgaggtatacaataattcaaaaagtcacacagaagaacttcaagcttataatgcaattagcagttc caatttatcttctaatcagaagtcaaagaagtttgaatttaaatctacagatatatacaatgatggatacgaaaccgtaa gctgcttcttaacgacagatcttaaaaaatattgtttaaattggaggtatgaatcaacagctttattcggtgatacttgt aatcagatatttgggttaaaggaattatttaattggctgcaccctcgccttgaaaagagtacaatatatgttggagatcc ttattgcccgccatcagatattgaacatttaccacttgatgaccatcctgattcaggattttatgttcataatcctaaag gaggaatagaagggttttgccaaaagttatggacactcatatctatcagtgcaatacatttagcagctgtcaaaatcggt gtaagagttactgcaatggttcaaggggataatcaagccatagctgttaccacaagagtacctaataattatgattataa agttaagaaagagattgtttataaagatgtggtaagattttttgattccttgagagaggtgatggatgatctgggtcatg agctcaaactaaatgaaactataataagtagtaaaatgtttatatatagcaaaaggatatactatgacggaagaatcctt cctcaggcattaaaagcattgtctagatgtgttttttggtctgaaacaatcatagatgagacaagatcagcatcctcaaa tctggctacatcgtttgcaaaggccattgagaatggctactcacctgtattgggatatgtatgctcaatcttcaaaaata tccaacagttgtatatagcgcttggaatgaatataaacccaactataacccaaaatattaaagatcaatatttcaggaat attcattggatgcaatatgcctccttaatccctgctagtgtcggaggatttaattatatggccatgtcaaggtgttttgt cagaaacattggagatcctacagtcgctgcgttagccgatattaaaagatttataaaagcaaatttgttagatcgaggtg tcctttacagaattatgaatcaagaaccaggcgagtcttcttttttagactgggcctcagatccctattcatgtaactta ccacaatctcaaaatataaccaccatgataaagaatataactgcaagaaatgtactacaggactcaccaaacccattact atctggattatttacaagtacaatgatagaagaggatgaggaattagctgagttcctaatggacaggagaataatcctcc caagagttgcacatgacattttagataattctcttactggaattaggaatgctatagctggtatgttggatacaacaaaa tcactaattcgagtagggataagcagaggaggattaacctataacttattaagaaagataagcaactatgatcttgtaca atatgagacacttagtaaaactttaagactaatagtcagtgacaagattaagtatgaagatatgtgctcagtagacctag ccatatcattaagacaaaaaatgtggatgcatttatcaggaggaagaatgataaatggacttgaaactccagatccttta gagttactgtctggagtaataataacaggatctgaacattgtaggatatgttattcaactgaaggtgaaagcccatatac atggatgtatttaccaggcaatcttaatataggatcagctgagacaggaatagcatcattaagggtcccttactttggat cagttacagatgagagatctgaagcacaattagggtatatcaaaaatctaagcaaaccagctaaggctgctataagaata gcaatgatatatacttgggcatttgggaatgacgaaatatcttggatggaagcatcacagattgcacaaacacgtgcaaa ctttacattggatagcttaaagattttgacaccagtgacaacatcaacaaatctatcacacaggttaaaagatactgcta ctcagatgaaattttctagtacatcacttattagagtaagcaggttcatcacaatatctaatgataatatgtctattaaa gaagcaaatgaaactaaagatacaaatcttatttatcaacaggtaatgttaacaggattaagtgtatttgaatatctatt taggttagaggagagtacaggacataaccctatggtcatgcatctacatatagaggatggatgttgtataaaagagagtt acaatgatgagcatatcaatccggagtctacattagagttaatcaaataccctgagagtaatgaatttatatatgataag gaccctttaaaggatatagatctatcaaaattaatggttataagagatcattcttatacaattgacatgaattactggga tgacacagatattgtacatgcaatatcaatatgtactgcagttacaatagcagatacaatgtcgcagctagatcgggata atcttaaggagctggttgtgattgcaaatgatgatgatattaacagtctgataactgaatttctgaccctagatatacta gtgtttctcaaaacatttggagggttactcgtgaatcaatttgcatataccctttatggattgaaaatagaaggaaggga tcccatttgggattatataatgagaacattaaaagacacctcacattcagtacttaaagtattatctaatgcactatctc atccaaaagtgtttaagagattttgggattgtggagttttgaatcctatttatggtcctaatactgctagtcaagatcaa gttaagcttgctctctcgatttgcgagtactccttggatctatttatgagagaatggttgaatggagcatcacttgagat ctatatctgtgatagtgacatggaaatagcaaatgacagaagacaagcatttctctcaagacatcttgcctttgtgtgtt gtttagcagagatagcatcttttggaccaaatttattaaatctaacatatctagagagacttgatgaattaaaacaatac ttagatctgaacatcaaagaagatcctactcttaaatatgtgcaagtatcaggactgttaattaaatcattcccctcaac tgttacgtatgtaaggaaaactgcgattaagtatctgaggattcgtggtattaatccgcctgaaacgattgaagattggg atcccatagaagatgagaatatcttagacaatattgttaaaactgtaaatgacaattgcagtgataatcaaaagagaaat aaaagtagttatttctggggattagctctaaagaattatcaagtcgtgaaaataagatccataacgagtgattctgaagt taatgaagcttcgaatgttactacacatggaatgacacttcctcagggaggaagttatctatcacatcagctgaggttat ttggagtaaacagtacaagttgtcttaaagctcttgaattatcacaaatcttaatgagggaagttaaaaaagataaagat agactctttttaggagaaggagcaggagctatgttagcatgttatgatgctacactcggtcctgcaataaattattataa ttctggtttaaatattacagatgtaattggtcaacgggaattaaaaatcttcccatcagaagtatcattagtaggtaaaa aactaggaaatgtaacacagattcttaatcgggtgagggtgttatttaatgggaatcccaattcaacatggataggaaat atggaatgtgagagtttaatatggagtgaattaaatgataagtcaattggtttagtacattgtgacatggagggagcgat aggcaaatcagaagaaactgttctacatgaacattatagtattattaggattacatatttaatcggggatgatgatgttg tcctagtatcaaaaattataccaactattactccgaattggtctaaaatactctatctatacaagttgtattggaaggat gtaagtgtagtgtcccttaaaacatccaatcctgcctcaacagagctttatttaatttcaaaagatgcttactgtactgt aatggaacccagtaatcttgttttatcaaaacttaaaaggatatcatcaatagaagaaaataatctattaaagtggataa tcttatcaaaaaggaagaataacgagtggttacagcatgaaatcaaagaaggagaaagggattatgggataatgaggcca tatcatacagcactgcaaatttttggattccaaattaacttaaatcacttagctagagaatttttatcaactcctgattt aaccaacattaataatataattcaaagttttacaagaacaattaaagatgttatgttcgaatgggtcaatatcactcatg acaataaaagacataaattaggaggaagatataatctattcccgcttaaaaataaggggaaattaagattattatcacga agattagtactaagctggatatcattatccttatcaaccagattactgacgggccgttttccagatgaaaaatttgaaaa tagggcacagaccggatatgtatcattggctgatattgatttagaatccttaaagttattatcaagaaatattgtcaaaa attacaaagaacacataggattaatatcatactggtttttgaccaaagaggtcaaaatactaatgaagcttataggagga gtcaaactactaggaattcctaaacagtacaaagagttagaggatcgatcatctcagggttatgaatatgataatgaatt tgatattgattaatacataaaaacataaaataaaacacctattcctcacccattcacttccaacaaaatgaaaagtaaga aaaacatgtaatatatatataccaaacagagtttttctcttgtttggt

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described embodiments. We claim all such modifications and variations that fall within the scope and spirit of the claims below. 

It is claimed:
 1. A recombinant chimeric bovine/human parainfluenza virus 3 (rB/HPIV3), comprising: a genome comprising, in a 3′ to 5′ order, a 3′ leader region, a BPIV3 N gene, a heterologous gene, BPIV3 P and M genes, HPIV3 F and HN genes, a BPIV3 L gene, and a 5′ trailer region; wherein the heterologous gene encodes one of: (a) a recombinant RSV G protein comprising a RSV G ectodomain, a BPIV3 HN transmembrane domain, and a BPIV3 HN cytoplasmic tail, wherein the recombinant RSV G protein comprises or consists of the amino acid sequence set forth as any one of SEQ ID NO: 31, 62, 64, 66, or 68, or an amino acid sequence at least 90% identical thereto; (b) a recombinant RSV G protein comprising a RSV G ectodomain, a HPIV3 HN transmembrane domain, and a HPIV3 HN cytoplasmic tail, wherein the recombinant RSV G protein comprises or consists of the amino acid sequence set forth as any one of SEQ ID NO: 70, 72, 74, 76, or 78, or an amino acid sequence at least 90% identical thereto; or (c) a recombinant RSV G protein comprising a RSV G ectodomain, a HPIV1 HN transmembrane domain, and a HPIV1 HN cytoplasmic tail, wherein the recombinant RSV G protein comprises or consists of the amino acid sequence set forth as any one of SEQ ID NO: 80, 82, 84, 86, or 88, or an amino acid sequence at least 90% identical thereto; wherein the HPIV3 HN gene encodes a HPIV3 HN protein comprising threonine and proline residues at positions 263 and 370 according to a reference HPIV3 HN protein sequence set forth as SEQ ID NO: 7; and wherein the recombinant B/HPIV3 is infectious, attenuated, and self-replicating.
 2. The rB/HPIV3 of claim 1, wherein the RSV G ectodomain comprises or consists of the amino acid sequence set forth as any one of SEQ ID NOs: 23, 48, 50, 52, or 54 or an amino acid sequence at least 90% identical thereto.
 3. The rB/HPIV3 of claim 1, wherein the BPIV3 HN transmembrane domain and cytoplasmic tail comprises or consists of the amino acid sequence set forth as SEQ ID NO: 29, or an amino acid sequence at least 90% identical thereto.
 4. The rB/HPIV3 of claim 1, wherein the HPIV3 HN transmembrane domain and cytoplasmic tail comprises or consists of the amino acid sequence set forth as SEQ ID NO: 57, or an amino acid sequence at least 90% identical thereto.
 5. The rB/HPIV3 of claim 1, wherein the HPIV1 HN transmembrane domain and cytoplasmic tail comprises or consists of the amino acid sequence set forth as SEQ ID NO: 60, or an amino acid sequence at least 90% identical thereto.
 6. The rB/HPIV3 of claim 1, wherein the recombinant RSV G protein comprising the RSV G ectodomain, the BPIV3 HN transmembrane domain, and the BPIV3 HN cytoplasmic tail comprises or consists of the amino acid sequence set forth as any one of SEQ ID NOs: 31, 62, 64, 66, or 68, or an amino acid sequence at least 90% identical thereto.
 7. The rB/HPIV3 of claim 1, wherein the recombinant RSV G protein comprising the RSV G ectodomain, the HPIV3 HN transmembrane domain, and the HPIV3 HN cytoplasmic tail comprises or consists of the amino acid sequence set forth as any one of SEQ ID NOs: 70, 72, 74, 76, or 78, or an amino acid sequence at least 90% identical thereto.
 8. The rB/HPIV3 of claim 1, wherein the recombinant RSV G protein comprising the RSV G ectodomain, the HPIV1 HN transmembrane domain, and the HPIV1 HN cytoplasmic tail comprises or consists of the amino acid sequence set forth as any one of SEQ ID NOs: 80, 82, 84, 86, or 88, or an amino acid sequence at least 90% identical thereto.
 9. The rB/HPIV3 of claim 1, wherein the RSV G ectodomain is from a human subtype A RSV or human subtype B RSV.
 10. The rB/HPIV3 of claim 1, wherein the RSV G protein is a wild-type RSV G protein from a human subtype A RSV or human subtype B RSV.
 11. The rB/HPIV3 of claim 1, wherein: the BPIV3 N gene encodes an N protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 1, or an amino acid sequence at least 90% identical thereto; the BPIV3 P gene encodes P, C, and V proteins comprising or consisting of the amino acid sequences set forth as SEQ ID NOs: 2, 3, and 4, respectively, or amino acid sequences at least 90% identical thereto; the BPIV3 M gene encodes an M protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 5, or an amino acid sequence at least 90% identical thereto; the HPIV3 F gene encodes an F protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 6, or an amino acid sequence at least 90% identical thereto; the HPIV3 HN gene encodes an HN protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 7, or an amino acid sequence at least 90% identical to SEQ ID NO: 7; and/or the BPIV3 L gene encodes an L protein comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 10, or an amino acid sequence at least 90% identical thereto.
 12. The rB/HPIV3 of claim 1, wherein the heterologous gene is codon-optimized for expression in human cells.
 13. The rB/HPIV3 of claim 1, wherein the genome comprises an antigenomic cDNA sequence set forth as any one of SEQ ID NOs:
 93. 14. The rB/HPIV3 of claim 1, wherein the rB/HPIV3 induces an immune response to RSV G protein, HPIV3 F protein, and HPIV3 HN protein.
 15. The rB/HPIV3 of claim 1, wherein the rB/HPIV3 induces an immune response that neutralizes RSV and HPIV3.
 16. A nucleic acid molecule comprising the nucleotide sequence of the genome of the rB/HPIV3 of claim 1, or an antigenomic cDNA or RNA sequence of the genome.
 17. A vector comprising the isolated nucleic acid molecule of claim
 16. 18. An isolated host cell comprising the nucleic acid molecule or vector of claim
 16. 19. A method of producing a rB/HPIV3, comprising: transfecting a permissive cell culture with the vector of claim 17; incubating the cell culture for a sufficient period of time to allow for viral replication; and purifying the replicated virus to produce the rB/HPIV3.
 20. A rB/HPIV3 produced by the method of claim
 19. 21. An immunogenic composition comprising the rB/HPIV3 of claim 1 and a pharmaceutically acceptable carrier.
 22. A method of eliciting an immune response to respiratory syncytial virus and human parainfluenza virus 3 in a subject comprising administering the immunogenic composition of claim 21 to the subject to generate the immune response.
 23. The method of claim 22, comprising intranasal administration of the immunogenic composition.
 24. The method of claim 22, wherein the subject is a human.
 25. The method of claim 22, wherein the subject is less than one year old.
 26. The method of claim 22, wherein the immune response is a protective immune response. 